SUMMARY The search for more effective treatment and prevention of tuberculosis has motivated the identification genes that are essential for the growth and survival of the causative agent, Mycobacterium tuberculosis (Mtb). These genes are potential new targets for developing and attenuated live vaccines. For example, targeting metabolite biosynthesis can prevent Mtb from making essential building blocks and generate auxotrophs that induce protective immunity, but do not cause disease. Riboflavin (vitamin B2) biosynthesis has been proposed as a drug target because putative biosynthetic genes have been shown as essential in multiple transposon screens, but Mtb is assumed to be unattainable as a riboflavin auxotroph and depend entirely on de novo synthesis in vivo because it lacks a homologue for a known bacterial riboflavin transporter. However, our critical review of the literature along with our preliminary data on inducible knockdown strains for riboflavin biosynthesis genes provide an opportunity to test our central hypothesis that Mtb takes up riboflavin via a noncanonical transport mechanism. We will test this hypothesis by identifying metabolites and genes that support Mtb growth when riboflavin biosynthesis is disrupted and determining whether riboflavin metabolites are limiting for Mtb growth in the host. If our hypotheses are correct, we expect to identify novel aspects of Mtb metabolite uptake and utilization and determine the feasibility of target riboflavin biosynthesis as drug targets in vivo, with potential impcats for the study of metabolite transporters in other intracellular pathogens.