Mycobacterium tuberculosis (Mtb) is an extremely successful pathogen that remains a key public health problem, infecting one-third of the human population and causing ~1.5 million deaths a year. The macrophage serves as a major host cell for Mtb, and the bacterium’s ability to turn the phagosome into a replicative niche is critical for the establishment and maintenance of chronic infection. This is dependent on Mtb sensing and responding appropriately to intraphagosomal signals, and the research proposed here builds on my discovery of chloride (Cl-) as a novel environmental cue that Mtb responds to during infection. I have further identified a Cl- channel in Mtb, and observed that a Mtb mutant deficient in this channel is severely attenuated for colonization in a murine infection model. My studies raise the novel concept that the sensing and/or maintenance of an appropriate balance of an abundant ion like Cl- can profoundly impact a pathogen’s ability to establish a successful infection, and suggest that this aspect of Mtb physiology represents a new target for the development of original and potentially potent tuberculosis treatment strategies synergistic with current approaches. Here, I seek to (i) elucidate the regulatory network governing Mtb’s sensing and response to Cl-,using both genetic and chemical biology approaches that exploit my Cl--responsive reporter Mtb strain, and (ii) examine how environmental cues, like Cl-, can impact on Mtb’s replication status, using genome-wide mutational analyses together with a novel replication reporter Mtb strain that I recently constructed.