DAP12 is an ITAM-containing adaptor molecule conveying activating properties to surface receptors on many cell types. We show here that DAP12 paradoxically down-modulates plasmacytoid dendritic cell (pDC) cytokine production in vivo during murine CMV (MCMV) infection. Higher levels of IFN-alphabeta and IL-12 were detected upon MCMV infection or CpG treatment in DAP12-deficient (DAP12(o)) mice as compared with wild-type (WT) mice. This resulted from altered homeostasis and enhanced responsiveness of pDCs in DAP12(o) animals. Increased numbers of pDCs were observed in the periphery of both naive and MCMV-infected DAP12(o) mice. A higher proportion of pDCs was activated in infected DAP12(o) mice, as demonstrated by intracellular staining using an optimized protocol for simultaneous detection of IFN-alpha and IFN-beta. The homeostasis of WT and DAP12(o) pDCs did not differ in mixed bone marrow chimeric mice. In addition, a similar efficiency of pDC differentiation was observed in vitro in Fms-like tyrosine kinase receptor 3 ligand cultures of WT and DAP12(o) bone marrow cells. This suggests that DAP12 signaling effects on pDC homeostasis are indirect. In contrast, in response to CpG, DAP12-mediated effects on both IL-12 and IFN-alphabeta production were intrinsic to the pDCs. However, in response to MCMV, only IL-12 but not IFN-alphabeta production was affected by pDC-intrinsic DAP12 signaling. Thus, DAP12 signaling in pDCs can mediate different regulatory effects on their functions, depending on the mechanisms of pDC activation. The potential implications of the regulation of pDC functions by DAP12 for promoting health over disease are discussed.