The treatment of tuberculosis (TB) is challenging – its etiological agent, Mycobacterium tuberculosis (Mtb), is intrinsically resistant to the majority of antibiotics normally effective against other bacterial pathogens, and the ones that demonstrate efficacy against Mtb require prolonged treatment durations to eliminate the pathogen from the host. Current treatment regimens result in toxic side-effects for many patients and often lead to patient non-compliance, which contributes to treatment failure and the development of drug-resistant TB. Consequently, there is a pressing need for the development of a shorter therapeutic regimen, as well as a better understanding of the factors limiting the effectiveness of current drugs. Transposon insertion sequencing (TnSeq) is a high-throughput methodology using next-generation-sequencing to quantify bacterial mutant phenotypes en masse. In this dissertation, we describe the use of TnSeq to address two different questions related to the problem of stress resistance in Mtb, in particular antibiotic resistance. The first problem was determining the functional role of mycobacterial acid resistance protease (MarP), a protein necessary for tolerance of not just acidic environments but also oxidative, detergent and antimicrobial stresses. Using a TnSeq-based genetic interaction screen, we identified genes that could be functionally related to marP. Our screen indicated that MarP had parallel functionality to multiple proteins involved in cell envelope biosynthesis and remodeling, suggesting a role for MarP in these processes. We also identified a few genes that could be functionally antagonistic to marP, possibly by promoting increased cell envelope permeability to substrates. The loss of marP also alleviated fitness defects resulting from mutations in the ESX-5 secretion system, the mycobactin biosynthesis pathway and rv0812, a previously uncharacterized gene. Characterization of rv0812 indicated that its main function was in para-aminobenzoic acid (PABA) biosynthesis, and that the loss of marP might promote activity of a bypass pathway to rv0812. In a second study, we screened for mutants with altered susceptibility to rifampicin, ethambutol, isoniazid, vancomycin and meropenem. Through TnSeq, we were able to identify and rank genes mediating antibiotic susceptibility in Mtb. Multiple cell envelope mutants were predicted to be strongly sensitive to the drugs tested, whereas inactivation of individual efflux pumps did not appear to contribute majorly to drug sensitivity. Together, the data from the two screens implicate the cell envelope to be a major determinant of Mtb resistance toward antibiotic and in vivo stresses, and suggest the existence of a functionally-linked cluster of cell envelope genes that could be synergistic targets supplementing current antibiotic regimens.