Best route to go Italy trip 2027

Tuscany-base in Siena otherwise you will want a car
Rome
Puglia
AC
Fly home from Naples

Direct flight to Rome on arrival would make more sense than Milan unless you have reason to stay in Milan

For your stay in Puglia, I recommend Polignano a Mare, a lovely seaside, small city.

Tuscany is closer to Rome than Milan, so I would fly to Rome instead of Milan, which is quite a distance from Florence (Tuscany). You don't seem to be interested in anything north of Tuscany, so Rome makes more sense. The majority of non stop flights from the NY area to Italy are to Rome.
If you plan to see Rome, then leave it for last before you fly back from Rome.
Alternatively you can also fly back from Naples, if you don't care to see Rome or if you plan to see Rome upon arrival.
There are non stop flights from Naples to Newark, NJ with United, but they are seasonal (late spring/summer only). So if you plan on that make sure they are available on the date you return.
From Tuscany to Puglia is a long way (like 500+ miles). If you go by car it's a good 9-10 hour drive. By train it is about the same, unless your departing point is Florence, which has high speed train. But still about 7 hours to Bari. There are flights to Bari, but only from Florence or Pisa.
To visit Tuscan towns, a rental car is definitely a plus, as many towns have limited bus service. Since you don't want to bother with Florence, Siena is the best base if you plan to use buses to visit Tuscany. If you rent a car, then you can stay anywhere.
A car in Puglia is also good to have. If you plan to rent a car for Tuscany, you might as well drive to Puglia and keep it for there too. I know it's a long drive, but the alternative is two separate rentals, which may not be convenient or even economical.

I suggest 4 nights in Tuscany, at least a week in Puglia, 4 nights for Sorrento/Amalfi Coast, at least 3 nights for Rome.

You could possibly rent a car for the whole trip and not bother with anything else. Obviously you should try not to have a car while in Rome. A car while in Sorrento/Amalfi Coast is also not necessary, so you could return it in Sorrento when you arrive, and go to Rome by train.
If you pick up a rental car at one location and return it to another there may be some one way drop off fees. They vary depending on the rental car company, and on the distance between the pick up and drop off location. I hear the highest one way fee will be less than 130€, but I don't know for sure. I've never paid more than 15€ in the recent past, but pick up and drop off were in the same region. I rented in Milan and returned in Florence a few times before Covid, but in those days they didn't charge me. I think they figured out to add that extra revenue now.

I appreciate all the advice :) We really don't want to rent a car. So thinking of skipping Amalfi Coast :( Flying to Rome yes 2 nights only we have been there before. Does anyone have advice on another city that is beautiful and charming from Tuscany(Florence) that we can stop for 2 nights before moving on to Puglia area(Bari) Trying to minimize travel I am also ok with train from Florence to Bari I believe there is a direct train I know its about 5-6 hrs. Suggestion please :)

Both Emilia-Romagna and the Veneto regions have "wineries and ... walkable towns and shops and Aperitivo" and lots of good trains. Also will be less heavily touristed.

Tuscany has other regions beat for medieval and Renaissance art and architecture (that's why I've spent several months there), but I always hope people will consider other areas if art is not their chief interest. And if they don't want to rent a car.

You can fly nonstop from either Bologna or Venice to Bari in less than an hour and a half. Direct train from Bologna to Bari takes 5 hours and 42 minutes.

We really don't want to rent a car. So thinking of skipping Amalfi
Coast :(

Of the 3 main locations you originally posted the AC is the one were you do not need or want a car, so I am not sure why you would skip?
Both Tuscany and Puglia benefit greatly from a car. Tuscany moreso than Puglia- trains and buses can get you more places in Puglia- not so much in Tuscany
Everywhere in Italy has wine, apertivo, good food, walkable towns and shopping.

Looks like you have about 2 weeks- 15 nights?
Why not stick to the south (you don't seem excited about time in Rome again)

United flies direct to Naples from EWR- starting early April

Fly in to Naples spend a few nights then train to Bari to start your time in Puglia.
After Puglia train back to the AC area- maybe starting in Salerno and work your way around the coast to Sorrento. Fly home from Naples. You might need to spend final night in Naples depends on what time- Think that flight leaves around 10 am.

You can easily fill 5-6 nights in the AC area and Puglia could use at least 6-7 nights

That's a lot less time spent on travel, more time being there.

Lovely towns or small cities with train stations in Emilia-Romagna that I have stayed in and enjoyed: Brisighella, Modena, Parma, and Ferrara. Parma has a nearby castle called Torrechiara that is one of my favorites due to its many frescoes rooms --- we took a bus there and a taxi back. Bologna is a city but has medieval and other nice walkable neighborhoods. Ravenna is more plain, but has some of the best mosaics in the world.

Thank you all for the adivice Gave me a lot to think about. :)

Sorry but why are you flying into Milano?

Every town has restaurants.

I think you might want to revise your plan a little.....so much hopping around...what month will this trip take place?

When is this? Sorry if I missed that.

United also has a non-stop to EWR from Bari. We are flying into Naples (Delta), 4 nights in Ischia before heading to Puglia with a 2 night stop in Matera. We are using Monopoli as our base in Puglia before flying back non- stop from Bari.

United also has a non-stop to EWR from Bari.

Oh wow! they sure do
Looks like EWR to Bari is M, W, F
Bari to EWR T, Th, Sat, Sun

Most be seasonal but I didn't check the start date for it
Very good to know

Thanks