Mary and her fixation on wishing Gary would propose to her after 10 years together. She's so completely dramatic about it.
During the "Man Or A Muppet" number, Gary and Walter sing about being at the crossroads of their lives and about how they need to choose whether they're "men or Muppets." In Gary's reflection, there is a Muppet version of him. In Walter's reflection, the audience expects to see the human version of him played by a live actor, and, apparently, get... Jim Parsons.
The fact that Parsons' role was kept secret until the film's release is pretty impressive.
Watch them when they're ushered out of the song: they all look fairy nonplussed, one of them is drinking something, another starts eating a bag of chips, and another is texting on her phone.
The Barber Shop Quartet scene where Beaker pops up with an axe, and the rest of the Muppets just stare at him awkwardly. The song they're doing is Nirvana's "Smells Like Teen Spirit". That was great, when they hold up towels with the lyrics Beaker's says "MEE MEE MEE MO" on it.
Scooter, who has never hosted anything before in his life, having to MC for Kermit when Kermit needs to go out for an emergency! Kermit advises him to do just what he does every time he's hosting: Imagine the Audience Naked.
Scooter: (stuttering and nervous) Y-y-y-y-YOU'RE ALL NAKED. (cut to shot of the audience in boxers and tank tops) ...well, almost.
Scooter introduced acts in the original show on occasion without a beat. He was probably nervous here because he hasn't done anything like that in decades and felt out of practice.
Beauregard has apparently been cleaning one closet of the Muppet Theater the entire time since the show ended, and never realized they all left!
The song "Me Party," mainly because of the restaurant patrons' complete and total obliviousness to Mary's song and dance.
Piggy sings this with a table of talking Muppet food in her dressing room including a lot of pastries. Why is the food there? 1.) Because she's lonely, and 2.) she's a pig!
The ending of "Life's a Happy Song," where, as soon as Gary, Mary, and Walter leave on the bus, the crowd lets out a relieved "They're gone!" and collapses.
Also during this song we get Willie Nelson singing "Life is full of Highs" and a couple of other people singing that "Life is a fillet of fish" to which Walter and Gary can only reply with... "Yes, yes it is".
The second performance has Tex Richman loudly remind everyone that they sang it already. Nobody seems to care.
Walter tells Kermit that he gave people the greatest gift in the world...
Walter: Uh, no, the one after that.
Kermit: Ice cream?
Walter: No, the other one.
Walter:[Excitedly] YES, LAUGHTER! THE THIRD GREATEST GIFT YOU CAN GIVE SOMEONE!
After you watch the telethon, you realize that it's very much exactly like how a regular episode of The Muppet Show would play out, save maybe for Jack Black being tied to a chair and doing nothing but complaining, and the money counter hanging throughout! It takes up nearly the same amount of time as a regular episode in the course of the film too, even though in-universe, it takes up two hours!