Something that’s always killed me about this is the redness of Sherlock’s upper lip after he rubs off the fake stache. I don’t know if it was intentional or just the result of several takes, but realistically you wouldn’t get that red after wiping some eyeliner off your skin.
The symbolicness of their mustaches is what makes it painful. John’s is real, Sherlock’s wasn’t. He fake-laughs to hide his desperation when he asks John if he’s keeping his. Because Sherlock is not. No more hiding. He is raw and exposed, and it hurts.