(1) Train Venice>Florence; (2) Rental Car

From Venice to Florence you have a choice of train companies, the national Trenitalia or the newer Italo. We used the latter from Venice to Rome last October and it was very nice. We have used Trenitalia as well, and it is just as comfortable. The Italo website is more user-friendly for new users.

http://www.italotreno.it/en

Both will give you a much better price if you book well in advance, but now is too early. I believe it is 120 days in advance for Trenitalia bookings; maybe more for Italo.

Make sure to use the correct stations--- Venezia Santa Lucia (Venezia S.L.) and Firenze Santa Maria Novella ( Firenze SMN).

Returning a car to the airport is no extra charge. "Picking Up" the car at an airport or train station will probably cost you more for the convenience of the location. So, rent in the city area and return at the airport (if the airport is your destination).

There is usually a schedule change for trains in mid June and a lot of the train prices won't be available until a month or so prior to that. They load the fastest trains first so you can start checking that schedule in April or May. Don't be too concerned if you find the routes are not available, it just means they haven't loaded that schedule yet. Keep checking. Book as soon as they become available to get the best prices.

Pick up and drop the car off at the Florence airport. It's quick and easy to do.

Donna

For car rental companies, many people here use either www.autoeurope.com or www.kemwel.com

TRAINS:
The Trenitalia website is:
www.trenitalia.com
The full (called BASE) price for a one way ticket on standard class is ~€45 one way per person. Higher class like business and executive cost more. I find the standard more than adequate, even better than Business class on an airplane. Tickets start selling online 120 days prior to the date. Substantial discounts are available if you secure a Supereconomy or Economy fare (30 to 70% off). Those deals are limited in number. Once sold, all you have left is the Base fare above. So it helps jumping on those early. There is a catch though. Tickets are specific to a specific date and train. Discounted tickets are not refundable or changeable if you miss the specific train you booked, and are not refundable, therefore you should buy them only if you are reasonably confident of your travel plans. If you want maximum flexibility, then you can pay full Base price and not worry about. There is no danger of a train being completely booked. There are one to two trains per hour between Venice and Florence, and they never sell out. So, if you pay full price, you can very confidently buy the ticket at the station on the day of departure or the day before. At most they sell out the standard class in the early morning trains, in which case you either buy a higher class (which never sells out) or wait for a later train. Trains in the middle of the day are often half empty in all classes.
Same principles apply with the other company (Italotreno), suggested above.

RENTAL CARS
Charlotte gave you above the name of the consolidator companies you should use (they are actually part of the same corporation after a merger years ago).
They work with the major companies operating in Italy (Avis-Budget, Europcar, Hertz, etc.). Check prices with both websites, as they vary in spite of being affiliate companies. In my experience renting cars thru them in Florence every year, I noticed that Europcar often offers the best deal in the above consolidators' website (I used both). I generally select the full no deductible insurance option. It's slightly more expensive but I like peace of mind and don't want to deal with headaches in case of accidents in Italy. Unfortunately I had my fair share of accidents there when I was living in Florence in my 20's.
You don't need to pick up the car at the airport. That will cost you more. You should pick up in the city and return it at the airport.
These are the pickup locations in the city (choose any)
CITY CENTER
Via Borgo Ognissanti (all companies: AvisBudget, Hertz, Europcar)
OUTSIDE CITY CENTER
Europcar: 2 Via Forlanini
Hertz: 53 via Del Sansovino
AvisBudget: none outside city center or airport.

The Borgo Ognissanti location is probably the closest to your hotel if you stay in the historical center. Be aware that from there you will not be able drive back to your Downtown hotel to pick up the luggage. Cars are not allowed in the historical center. Therefore, when you go to the rental place, go with your luggage and spouse ready to leave town. From Borgo Ognissanti you can only drive west toward Porta Al Prato, turn right on Viale Rosselli and go to your destination. If you select a pick up at a location outside the city center, the same principle apply. You cannot drive back into the city center to your city center hotel.
I generally use Europcar and pick up on Via Forlanini (located between the city center and the airport in the Novoli district). The reason is that on Borgo Ognissanti there are often long lines of tourists trying to rent a car, especially in summer. Made that mistake once (2 hours in line to pick up my car), not doing it again.

You will certainly need a taxi to reach Via Forlanini (Europcar) or Via Del Sansovino (Hertz). They are not walking distance from the city center.

Avis-Budget on via Borgo Ognissanti has rarely any lines, simply because they always have higher rental rates (I rented from them too). They only have that location and the airport (the airport has all companies).

The only cautionary thing I've heard about picking a car up in the city is you need to be aware of where those ZTL zones are. Cars need special permits to travel in those zones, and rental cars apparently don't have them. They have traffic cameras all over the place, and if they catch you, they'll fine the rental company, which will in turn fine you. I haven't experienced this personally, but it's been mentioned to me many times (I, too, will be picking up a car in Florence for my Tuscany excursion).

Jonathan,

Also note that for driving in Italy, you'll require the compulsory International Driver's Permit, which is used in conjunction with your home D.L. As the others mentioned, be very careful to avoid the dreaded ZTL (limited traffic) areas which now exist in many Italian towns and cities. Florence is just about saturated with automated ZTL cameras, and EACH pass through one will result in a hefty fine, which you won't know about until several months after you return home. Roberto provided an excellent description on how to avoid those.