The mechanisms of protective immunity to tuberculosis remain poorly understood in humans. A whole-blood infection model that employs a luminescent readout was used to analyze the role of T cells in control of mycobacterial infection. Control of mycobacterial growth in blood from healthy tuberculin-positive individuals was shown to be mediated predominantly by CD4(+) T cells. Comparison of age-matched cohorts of human immunodeficiency virus (HIV)-infected and -uninfected children from South Africa demonstrated an association between low CD4 cell counts, low interferon (IFN)-gamma production, and impaired ability to regulate growth of Mycobacterium bovis bacille Calmette-Guérin in blood from HIV-infected children. Impaired control of infection was not reconstituted by the addition of exogenous IFN-gamma. The whole-blood assay provides an important tool for monitoring and dissecting of human immune responses to mycobacterial infection.