Dendritic cells (DC) produce interleukin-12 (IL-12) in response to Toll-like receptor (TLR) activation. Two major TLR signaling pathways participate in the response to pathogens: the nuclear factor-κB (NF-κB)–dependent pathway leading to inflammatory cytokine secretion including IL-12 and the interferon (IFN)-dependent pathway inducing type I IFN and IFN-regulated genes. Here we show that the two pathways cooperate and are likely both necessary for inducing an optimal response to pathogens. R-848/Resiquimod (TLR7 ligand in the mouse and TLR7/8 ligand in human) synergized with poly(I:C) (TLR3 ligand) or lipopolysaccharide (LPS; TLR4 ligand) in inducing high levels of bioactive IL-12p70 secretion and IFN-β mRNA accumulation by mouse bone marrow–derived DC (BM-DC). Strikingly, IL-12p70 but not IL-12p40 secretion was strongly reduced in BM-DC from STAT1^−/− and IFNAR^−/− mice. STAT1 tyrosine-phosphorylation, IL-12p35, and IFN-β mRNA accumulation were strongly inhibited in IFNAR^−/− BM-DC activated with the TLR ligand combinations. Similar observation were obtained in human TLR8-expressing monocyte-derived DC (moDC) using neutralizing anti-IFNAR2 antibodies, although results also pointed to a possible involvement of IFN-λ1 (also known as IL-29). This suggests that TLR engagement on DC induces endogenous IFNs that further synergize with the NF-κB pathway for optimal IL-12p70 secretion. Moreover, analysis of interferon regulatory factors (IRF) regulation in moDC suggests a role for IRF7/8 in mediating IRF3-independent type I IFN and possibly IL-12p35 synthesis in response to TLR7/8.