Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the Δ-dosR, Δ-dosS, and Δ-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although Δ-dosR and Δ-dosT grew comparably to wild-type Mtb, Δ-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. Δ-dosS retained the ability to induce DosR. The Δ-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of Δ-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of Δ-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.