Rationale: Mycobacterium tuberculosis (Mtb) has a devastating impact on global health. Mtb is the causative agent of human pulmonary tuberculosis (TB), which claims 1.4 million lives per annum. During infection, differentially culturable bacilli (DCB) that fail to produce colonies on solid media but grow in liquid media supplemented with resuscitation-promoting factor (Rpf) proteins are formed. These Rpf-dependent mycobacteria are a major barrier to developing improved TB treatments and diagnostic tools. Objectives: To evaluate whether exposure to nitric oxide (NO), a prominent host defense molecule, drives the generation of Rpf-dependent DCB. Methods: A new NO donor (ND) for sustained NO release was synthesized. Growth assays, flow cytometry analysis, and global transcriptional profiling, were used to characterize ND-treated Mtb and Mycobacterium bovis BCG in vitro. Mycobacterial DCB phenotypes were also investigated after infection of THP-1 human cells, which were activated with retinoic acid and vitamin D3. Measurements and Main Results: DCB were generated after exposure to ND and during infection of activated THP-1 cells. Resuscitation of these DCB was severely impaired by a specific Rpf inhibitor (3-nitro-4-thiocyanato-phenyl)-phenyl-methanone. Global transcriptomic analyses revealed the down-regulation of three (rpfA, rpfB and rpfE) of the five genes coding for Rpf proteins. Conclusion: A new model for investigation of the transition of Mtb into an Rpf-dependent differentially culturable state is presented. The system reveals mechanistic insights into the generation of Rpf-dependent Mtb during TB disease and demonstrates the role of NO in this process.