Germline mutations in five autosomal genes involved in interleukin (IL)-12–dependent, interferon (IFN)-γ–mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)–MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-κB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell–dependent IL-12 production, resulting in defective IFN-γ secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-κB/c-Rel–mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-κB activators, such as tumor necrosis factor-α, IL-1β, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell– and CD40L-triggered, CD40-, and NEMO/NF-κB/c-Rel–mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.