This study investigates the biochemical environment of *Mycobacterium tuberculosis* (MTB) within macrophage phagosomes, focusing on the transcriptional responses of MTB in wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with MTB in standard broth culture and under diverse conditions designed to simulate the phagosomal environment. Key findings include: 1. **Genes Induced by Intraphagosomal Residence**: Genes expressed differently due to intraphagosomal residence include those involved in interferon γ- and NO-induced responses, iron scavenging, conversion to anaerobic respiration, and dormancy regulation. 2. **Fatty Acid Metabolism**: Induction of genes involved in fatty acid activation and β-oxidation suggests that fatty acids provide carbon and energy for MTB. 3. **Cell Envelope Damage and Repair**: σF-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification indicate damage and repair of the cell envelope. 4. **Sentinel Genes**: Sentinel genes within the intraphagosomal transcriptome were similarly induced by MTB in the lungs of mice, indicating a consistent response to the phagosomal environment. 5. **Phagosomal Environment**: The microbial transcriptome reveals a nitrosative, oxidative, functionally hypoxic, carbohydrate-poor environment capable of perturbing the pathogen’s cell envelope. The study provides insights into how MTB adapts to the harsh conditions within macrophage phagosomes, highlighting the importance of NO and other stressors in shaping the phagosomal environment.This study investigates the biochemical environment of *Mycobacterium tuberculosis* (MTB) within macrophage phagosomes, focusing on the transcriptional responses of MTB in wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with MTB in standard broth culture and under diverse conditions designed to simulate the phagosomal environment. Key findings include: 1. **Genes Induced by Intraphagosomal Residence**: Genes expressed differently due to intraphagosomal residence include those involved in interferon γ- and NO-induced responses, iron scavenging, conversion to anaerobic respiration, and dormancy regulation. 2. **Fatty Acid Metabolism**: Induction of genes involved in fatty acid activation and β-oxidation suggests that fatty acids provide carbon and energy for MTB. 3. **Cell Envelope Damage and Repair**: σF-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification indicate damage and repair of the cell envelope. 4. **Sentinel Genes**: Sentinel genes within the intraphagosomal transcriptome were similarly induced by MTB in the lungs of mice, indicating a consistent response to the phagosomal environment. 5. **Phagosomal Environment**: The microbial transcriptome reveals a nitrosative, oxidative, functionally hypoxic, carbohydrate-poor environment capable of perturbing the pathogen’s cell envelope. The study provides insights into how MTB adapts to the harsh conditions within macrophage phagosomes, highlighting the importance of NO and other stressors in shaping the phagosomal environment.