Since the first description of cellular autofluorescence over a century ago, we have now come to appreciate that autofluorescence should not be discarded as a biological artifact but embraced as a biological phenomenon with potentially important cellular relevance. Indeed, cellular and tissue autofluorescence has been attributed to a spectrum of unrelated molecules such as porphyrins, vitamins (vitamin A, riboflavin, thiamine), structural proteins, lipofuscin and ceroid pigments. We have recently shown that freshly isolated epithelial cancer stem cells (CSCs) bear autofluorescent vesicles in the cytoplasm. Our studies definitively prove that riboflavin and not lipofuscin is the source of autofluorescence in CSCs as the inhibition of ATP and not autophagy eliminates CSC autofluorescence, that the ATP-dependent transporter ABCG2, for which riboflavin is a substrate, is overexpressed in autofluorescent CSCs and co-localizes with the membrane of intracellular autofluorescent vesicles, the ABCG2-specific inhibitor Fumitremorgin C reversibly eliminates CSC autofluorescence, riboflavin is a substrate for ABCG2, and only the addition of riboflavin to vitamin-deprived CSC cultures is capable of restoring autofluorescence. Thus, the sum of these data unequivocally supports the conclusion that the source of CSC autofluorescence is the vitamin riboflavin.