Vitamin B2 (riboflavin) is essential for metabolic functions and is synthesized by many bacteria, yeast and plants, but not by mammals and other animals, which must acquire it from the diet. In mammals, modified pyrimidine intermediates from the microbial biosynthesis of riboflavin are recognized as signature biomarkers of microbial infection. This recognition occurs by specialized lymphocytes known as Mucosal Associated Invariant T (MAIT) cells. The Major Histocompatibility class I-like antigen presenting molecule, MR1, captures these pyrimidine intermediates, but only after their condensation with small molecules derived from glycolysis and other metabolic pathways to form short-lived antigens. The resulting MR1-Ag complexes are recognized by MAIT cell antigen receptors (alphabeta TCRs) and the subsequent MAIT cell immune responses are thought to protect the host from pathogens at mucosal surfaces. Here we review our understanding of how these novel antigens are generated and discuss their interactions with MR1 and MAIT TCRs.