Sienna, Gubbio, Todi, Spello or Orvieto

To me, Orvieto stands out above the rest. The duomo is splendid, both outside and in. If you go, don't miss the Luca Signorelli frescoes in a side chapel. Additional cost, but very worth it. Orvieto has, as well, a small Etruscan museum, some underground sights, charming little side streets, and good restaurants. Spello is pretty small. Not sure there is enough there for even 2 nights.

I second Orvieto, but unless you have a car for day trips four nights might be a little too long. Maybe two nights Orvieto and two nights Assisi (with a day trip to Spello on the train)? Or Sienna instead of Assisi if you liked it better.

Orvieto can be a nice day trip from Rome; if you spend some time in Assisi, Spello is only a few kilometers away. Gubbio is also within reach from Assisi. Todi is beautiful but can get quite crowded during the day. Siena has more to offer than the other cities on your list, in my opinion, but keep in mind that using public transportation (especially trains) from Siena or Assisi involves a few extra minutes to get to the train stations. Buses might be more convenient, a car even more so.

I really like Siena and Orvieto. I was pleasantly surprised by the sights they offered - both are a little bigger than the average hill town. I haven't been to Gubbio, Todi or Spello so can't give you a good opinion on those.

I would stay all 3 nights in Siena, and 1 night in Orvieto.