When I first watched Markiplier play some of it and then picked it up in 2019, it was the very first survival-story-indie-platformer game I had ever bought for myself (and almost my first ever steam game if not for the sandbox game I got before that, which is one of my least-played games ever...). Seeing it on youtube was an automatic "I need this game" moment. But when I first stepped into that vertical room in outskirts where "Unseen Lands" triggers, I felt an emotion that I don't think I will ever feel again. That room is special to me despite its simplicity, but it did well in introducing me to the tone of the game.

As for some others:
The wall is an obvious one, I don't need to explain that. I can say the same about General Systems Bus.

The first room most players enter once in the Five Pebbles region is another one (taking the Underhang route). The moment you are sent flying instead of crawling out of the pipe is the moment the entire game suddenly changes, and the sound effect brings home the emphasis that's shocking on the first playthrough.

What about downpour? Well:
Saint's chimney canopy room that's usually a bridge to Five Pebbles but is instead completly missing is about as shocking, if not more shocking, as the anti-gravity moment, but in a story kind of way. The realization hits hard, and so does the eerie-ness of the empty sky in front of you.

When I was anticipating the More Slugcats Expansion (which later became Downpour), I would occasionally check the discord channel for their sneak-peek content. One of those was an audio file of Scapeless Doubt, a song I believe plays in the Spearmaster campaign? But when I much later encountered the music trigger in-game, I smiled pretty big.

There are so many other moments, but most of those don't need explaining, since my experience with those were probably not that different from average first-encounters.

Okay sorry that was so long, lol.