Handicap accommodations in Italy

That will vary from case to case.
All buildings, including hotels, must abide by certain regulation codes to facilitate people with disabilities. However that applies to new constructions. Many old building, including hotels, predate any such regulations, and such reconfiguration were not possible. So for example there will be hotels in buildings with no elevators. Same applies to various sites, like museums, and other historical sites. For example if one wants to go on top of the Duomo in Florence or the leaning tower in Pisa, there are no elevators, only stairs. So you will need to see each individual situation at each specific sight.
Going in front of a long line, will not be a problem. I am sure that will be the case in many places in Italy too.

As Roberto said. Keep in mind that many museums have elevators, some require that you be escorted to them by staff (who have a key).

Also, many restaurants and bars have restrooms down a flight of stairs.

If you need assistance at train stations, there is a list of which accommodations are available; most major stations have staff assistance if you need it.

Hi Kris,
My info is from 2006 and probably different from your situation, but I took my Mom to Italy and she used her wheelchair almost full time. We made reservations ahead of time at the Vatican Museum for instance, and we were there when they opened and they took us behind ropes, to ramps and such, very helpful. We went to Rome, Tuscany & the CT by car & Venice. It seemed like people were surprised to see us in the main tourist spots with the chair, but most people were helpful, especially the vaporetto crews.

If the number of stair steps is important to you, confirm details with your hotels when booking, because simply having an elevator is not always enough info. Some elevators start a half-flight or full flight of stairs up from street level and occasionally have service issues (I'm thinking of budget hotels in Rome, where each user must close the ancient elevator doors properly). A hotel can also have some stairs "between floors" if they've pieced different annexes together.

Transport options in Rome are similar to in Paris, but the Metro routes are less extensive and the buses run quite full. Taxis are affordable and accessed at a taxi stand or by phone call or app. For 5 people, you probably need a larger vehicle. Florence's sightseeing center is compact, so fewer tourists use taxi and bus options, but they're there.

For train boarding assistance, see http://www.trenitalia.com/tcom-en/Purchase/Info-&-Contacts/Disabled-passengers. Rome-Florence is served by the newest trains: http://www.trenitalia.com/tcom-en/Frecce/Frecciarossa. Florence SMN station has all the important things on one level http://www.firenzesantamarianovella.it/en/map/ and Roma Termini does not but has escaltors and elevators: http://www.romatermini.com/en/.

You will certainly have priority over fit people and for many sites if you have a handicapped card, it might be free (do ask) for you and another person to help you. My Italian friend who has a degree of invalidity is given this right quite often.

Florence is rather flat, so this must be easier for you than hilly towns such as Siena. Another problem is that these are medieval cities and most buildings are not fit for lifts, so do ask before booking your accommodation, in big museums, there might be facilities for handicapped people ( lift for handicapped only). It might also be difficult to sit if you get tired easily, for example at the Offices.

Thank you for all the great input, I am looking forward to exploring Italy and we will deal with whatever comes along.