ABSTRACTMultidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains ofMycobacterium tuberculosis. We reasoned that auxotrophic MDR mutants ofM. tuberculosiswould provide a safe means for studying MDRM. tuberculosiswithout the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants ofM. tuberculosiswere generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations inkatG, which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containingkatG. TheseM. tuberculosisMDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates forM. tuberculosisbiology studies and drug screening outside the BSL3 facility.IMPORTANCEElimination ofMycobacterium tuberculosis, the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistantM. tuberculosisas well as uncovering novel pathways that lead toM. tuberculosisdeath. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research onM. tuberculosis, we have generated drug-susceptible and drug-resistant triple auxotrophic strains ofM. tuberculosissuitable for use in a BSL2 laboratory. These strains originate from a double auxotrophicM. tuberculosisstrain, H37Rv ΔpanCDΔleuCD, which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third auxotrophy (methionine or arginine) was introduced via deletion ofmetAorargB, respectively, sinceM. tuberculosisΔmetAandM. tuberculosisΔargBare unable to survive amino acid auxotrophy and infect their host. The resulting triple auxotrophicM. tuberculosisstrains retained characteristics ofM. tuberculosisrelevant for most types of investigations.