The IL-12/IFN-γ axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-γ. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guérin (BCG) alone, or BCG plus IL-12 or IFN-γ, measuring IFN-γ and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-κB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor β1 chain (IL-12Rβ1), IFN-γR1, IFN-γR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-γ. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-γ production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-γ axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-γ axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.