Mycobacterium tuberculosis (Mtb) is the world’s leading infectious cause of mortality. Despite the overwhelming data supporting the critical role of cellular immunity, little is known of the early microbial and immune cell interactions and whether human macrophages can be activated to express anti-Mtb activity. We report the reconstitution of an in vitro system whereby human macrophages express anti-Mtb activity only in coculture with PBL and with IFN-γ. Omission of IFN-γ in the cocultures or Mtb lysate/IFN-γ-primed lymphocytes was associated with high growth of Mtb, high IL-10 and IL-12 p40, nearly undetectable IL-12 p70 levels, and the highest percentages of CD4 and CD8 T cells. In contrast, IFN-γ treatment of cocultures containing Mtb lysate/IFN-γ-primed PBL reduced bacilli count by∼ 2.5 log, decreased the production of IL-10 by 5.7-fold, increased IL-12 p70 by∼ 50-fold, and reduced the percentages of CD4 and CD8 T cells. Activation of anti-Mtb activity was time and dose dependent. At 2000 U/ml of IFN-γ, bactericidal activity was achieved (10-fold reduction from initial inoculum). Anti-Mtb activity against several strains of M. tuberculosis (H37Ra and H37Rv, and C, a clinical isolate) was observed and was associated with expression of inducible nitric oxide synthase. These data suggest that induction of human macrophage anti-Mtb activity required dual signaling from PBL and IFN-γ. Thus, the development of an in vitro human system may greatly facilitate studies to delineate immune cells, cytokines, and effector functions/genes critical in controlling Mtb. Defining the mechanisms may also provide novel treatment strategies for tuberculosis.