Batch cultures have predominately been used for the study of physiology and gene expression in mycobacteria. This chapter describes the assembly of chemostats and the methodology that is being used for growing Mycobacterium tuberculosis in continuous culture, which provides the greatest control over experimental conditions. It is difficult to determine the underlying genetic changes that enable M. tuberculosis to adapt to the host environment, but in vitro experiments aid the interpretation of gene expression profiles of the bacillus in vivo. Selecting relevant host conditions for study presents a major challenge. Oxygen availability has been identified as an important environmental stimulus and is a simple parameter to adjust and monitor. Described here are continuous culture methods to determine the response of M. tuberculosis to low oxygen environments.