Cellular FLICE inhibitory protein (c-FLIP) specifically inhibits caspase-8 and suppresses death receptor-induced apoptosis. c-FLIP has also been reported to transmit activation signals. Here we report a novel function of c-FLIP involving inhibition of myeloid cell activation through antagonizing the selective innate signaling pathway. We found that conditional knockout of c-FLIP in dendritic cells (DCs) led to neutrophilia and splenomegaly. Peripheral dendritic cell populations, including CD11b+ conventional DC (cDC), CD8+ cDC, and plasmacytoid DC (pDC), were not affected by c-FLIP deficiency. We also found that c-FLIP-knockout cDC, pDC, and bone marrow-derived DC (BMDC) displayed enhanced production of TNF-, IL-2, or G-CSF in response to stimulation of TLR4, TLR2, and dectin-1. Consistent with the ability of c-FLIP to inhibit the activation of p38 MAPK, the enhanced activation of c-FLIP-deficient BMDCs could be partly linked to an elevated activation of p38 MAPK after engagement of innate receptors. Increased activation was also found in c-FLIP+/- macrophages. In addition, the increased activation in c-FLIP-deficient dendritic cells was independent of caspase-8. Our results reveal a novel inhibitory role of c-FLIP in myeloid cell activation and demonstrate the unexpected anti-inflammatory activity of c-FLIP. In addition, our observations suggest that cancer therapy target c-FLIP downregulation may facilitate DC activation and increase T cell immunity.