It depends on how many differences there are, or how many you assume there are, not on how many items you are comparing.
As you can see, the number of items being compared doesn't change anything about these sentences (aside from the actual number used).
However, it's also true that the greater the number of items you are comparing, the more likely it is there will be more than just a single difference between all of them.
That being said, it's often known that there are multiple things that are different between one thing and another—and it can either be between two things or more than two things.
But even knowing that there are, technically, multiple differences, unless the person asking the question is doing so as a kind of quiz or they are deliberately calling attention to the plural number of differences, it's still often idiomatic to phrase it in the singular.
For example, let's say I'm shopping at a store for a new coffee maker. I see ten different models on display, and somebody comes up to me:
"Can I help you with something?"
It might be perfectly obvious that there are many differences between the ten coffee makers. Nonetheless, idiomatically, I could answer in one of these ways:
"Yes, can you tell me what the difference is between these?"
"Yes, can you tell me how these are different?"
(Note that the second response uses a different form of grammar; in How are these different, different is neither singular nor plural, because it's an adjective rather than a noun.)
On the other hand, while the response in the singular is fine idiomatically, so too would be the following more technically correct responses:
"Yes, can you tell me what the differences are between these?"
"Yes, could you please describe all of the differences between these?"
In short, for routine casual and imprecise conversation, you can use difference, even if you know there isn't just a single difference.
For precise conversation or description, where the exact number is important, it's not the number of items being compared that determines what's said, but the number of actual or assumed differences that are present.