Tuberculosis (TB) is characterized by lifetime persistence of Mycobacterium tuberculosis. Despite the induction of a vigorous host immune response that curtails disease progression in the majority of cases, the organism is not eliminated. Subsequent immunosuppression can lead to reactivation after a prolonged period of clinical latency. Thus, while it is clear that protective immune mechanisms are engaged during M. tuberculosis infection, it also appears that the pathogen has evolved effective countermechanisms. Genetic studies with animal infection models and with patients have revealed a key role for the cytokine gamma interferon (IFN-gamma) in resistance to TB. IFN-gamma activates a large number of antimicrobial pathways. Three of these IFN-gamma-dependent mechanisms have been implicated in defense against M. tuberculosis: inducible nitric oxide synthase (iNOS), phagosome oxidase (phox), and the phagosome-associated GTPase LRG-47. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis. Using this approach we have identified three M. tuberculosis genes that are essential for progressive M. tuberculosis growth and rapid lethality in iNOS-deficient mice but not in IFN-gamma-deficient mice. We propose that these genes are involved in pathways that allow M. tuberculosis to counter IFN-gamma-dependent immune mechanisms other than iNOS.