ABSTRACTMycobacterium tuberculosis(Mtb), the causative agent of tuberculosis, is one of the oldest and most successful pathogens in the world. Diverse selective pressures encountered within host cells have directed the evolution of unique phenotypic traits, resulting in the remarkable evolutionary success of this largely obligate pathogen. Despite centuries of study, the genetic repertoire utilized byMtbto drive virulence and host immune evasion remains to be fully understood. Various genetic approaches have been and continue to be developed to tackle the challenges of functional gene annotation and validation in an intractable organism such asMtb.In vitroandex vivosystems remain the primary approaches to generate and confirm hypotheses that drive a general understanding of mycobacteria biology. However, it remains of great importance to characterize genetic requirements for successful infection within a host system asin vitroandex vivostudies fail to fully replicate the complex microenvironment experienced byMtb.In this review, we evaluate the employment of the mycobacterial genetic toolkit to probe the host-pathogen interface by surveying the current state of mycobacterial genetic studies within host systems, with a major focus on the murine model. Specifically, we discuss the different ways that these tools have been utilized to examine various aspects of infection, including bacterial survival/virulence, bacterial evasion of host immunity, and development of novel antibacterial/vaccine strategies.