Natural products frequently inspire chemical efforts to unravel complex mechanisms in biology, health and disease. Over the past decade, we have investigated chemistry related to the bacterial synthesis of riboflavin (vitamin B2) and discovered unique uracil derivatives that potently activate a relatively new class of mucosal T lymphocytes known as MAIT cells [1]. These innate-like immune cells respond to bacterial, fungal and viral infections (including M. Tuberculosis, HIV, influenza, SARS-CoV-2) and also regulate tissue repair, respiratory and inflammatory bowel diseases, graft-vs-host disease, gastritis, and cancers. Unlike other T cells, MAIT cells are not activated by peptide or lipid ligands but instead are activated by small heterocyclic organic molecules. This presentation will briefly summarize the discovery of bacterial ligands formed through condensation of a riboflavin biosynthetic intermediate and metabolic byproducts of microbial and mammalian glycolysis. It will describe their development into fluorescent and immunological reagents for detecting and characterizing MAIT cells in tissues, and demonstrate how chemical biology has helped to unravel molecular mechanisms of antigen capture and T cell activation to enable potential therapeutic applications for infections and cancers.