A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe3+) form, whereas DosT exists in the O2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O2, NO, and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO, that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence.