Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-gamma) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-gamma. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-gamma message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-gamma responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-gamma responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R(2) = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-gamma by 39% compared to the level with antigen alone. Increased production of IFN-gamma induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-gamma-based assays might improve TB diagnosis in those populations at highest risk for TB.