Studies of human tuberculosis have been hampered by the lack of an in vitro model that recapitulates all the features of a natural infection with Mycobacterium tuberculosis. This paper describes a unique combination of in vitro culture conditions that enables the generation of human monocyte-derived macrophages (MDMs) that can survive infection with M. tuberculosis and control its replication for more than 2 weeks. The new protocol involves monocyte culture for 2 weeks at 10% oxygen in 40% human plasma containing the cytokines GM-CSF and TNF, with the medium replaced every 3–4 days, followed by 'activation' with interferon-γ for 2 days prior to infection. The authors showed that culture in fetal bovine serum or with M-CSF (two additives that are commonly used for the culture of human monocytes) led to the generation of MDMs that were unable to control M. tuberculosis replication and died.