Mycobacterium tuberculosis resides within the phagocytes of its host. It ensures its continued survival through arresting the normal maturation of its phagosome, which is retained within the early endosomal system of the macrophage. Although individual bacterial components have been shown to modulate phagosome biogenesis, the mechanism(s) active in live, intact bacteria remain elusive. We have developed a genetic screen that facilitates the isolation of mutants defective in arresting the maturation of their phagosomes. Macrophages were incubated with iron-dextran that was chased into lysosomes. The cells were subsequently infected with M. tuberculosis from a library of transposon-mutagenized bacteria. After four rounds of enrichment, the majority of mutants isolated were unable to prevent acidification of their phagosomes and were attenuated for intracellular survival. The genes affected range in function from those with no known homologues to putative transporters and lipid synthesis enzymes. Further characterization of these bacteria is needed. In addition to clarifying the processes active in modulation of phagosome biogenesis by M. tuberculosis, this screen may be applicable to other pathogens that restrict the maturation of their phagosome.