Actually, you’re not really asking about FLAC specifically per se’, you’re asking about all lossless audio in general, considering the question of whether or not you can actually tell the difference applies to all lossless audio, not just FLAC.
It’s actually easy to tell the difference between .pngs and .jpgs with most pictures, because .jpgs have compression artifacts and fuzzy places that .pngs don’t have. But with FLAC and MP3s, or lossless and lossy considering that’s what we’re really talking about here, some songs you can tell a bit of difference, and some you can’t.
Rob is right that in most cases, when the sounds of life are going on around you, you can’t really tell the difference. But if you’re in a quiet environment or listening on good headphones, sometimes you can tell on some of the lower frequencies and even more on the higher frequencies.
Another place it comes into play is if you’re going to burn something to a CD. If you burn to a CD from a lossy file, it’s like a third generation copy, whereas burning something from a lossless file gives you a second generation copy so it’s a bit better there too.
As far as MP3s go, I actually don’t have many MP3s. The only MP3s I really have are either podcasts, or various recordings. All of my music is in OGG format, which is the open source equivalent of MP3.
So basically, in most situations most people won’t be able to tell the difference between a lossy and lossless audio file, but there are some situations where it’s nice, whether it’s proprietary or open source.