The ability of all pathogens to survive within the host is key to their success in establishing disease. Environmental conditions that affect the growth of a pathogen in the host include nutrient status, environmental pH, oxygen availability, and host defences. Studying the response of Mycobacterium tuberculosis (M. tuberculosis) exposed to these relevant host conditions in vitro will further increase our understanding of how these environments have an impact on the molecular mechanisms M. tuberculosis adopts to combat the effects of external influences such as antimycobacterials. The methods presented here are used to investigate the effect of environmental factors on the development of drug-resistant M. tuberculosis. Cultures grown under controlled conditions in continuous culture are sampled and the frequency with which resistant mutants develop are determined. These studies provide data that aid our understanding of the complex interaction between the host environment and invading bacterium that allow resistant strains to develop and continue to cause disease.