IL-12 is such a pivotal cytokine that it has been called the third signal for T cell activation, TCR engagement being the first and costimulation being the second. It has been generally viewed that the resident CD8(+) dendritic cell (DC) subset is the predominant IL-12-producing cell type. In this study, we found, although this is so under steady state conditions, under inflammatory conditions monocyte-derived DC (mDC) became a major cell type producing IL-12. Depletion of either type of DC resulted in reduced production of IL-12 in vivo. For CD8(+) DC, IL-12 production could be stimulated by various pathways viz. signaling through MyD88, Trif, or nucleotide-binding oligomerization domain (Nod)-like receptors. In contrast, for mDC, IL-12 production was mainly dependent on MyD88 signaling. Thus, conventional DCs and mDCs use different pathways to regulate IL-12 production. The Journal of Immunology, 2010, 185: 2125-2133.