TLR4 ligation can activate both the MyD88 and the Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) signaling route. Whereas MyD88 is generally recognized as a universal adaptor for pro-inflammatory responses, TRIF is mainly thought to contribute to specific type I IFN responses. Here, we investigated the contribution of both MyD88 and TRIF to TLR4-mediated pro-inflammatory dendritic cell (DC) differentiation in human. Pro-inflammatory cytokine induction was strongly decreased in monophosphoryl lipid A- and LPS-matured monocyte-derived DCs when either MyD88 or TRIF were down-regulated by small interfering RNA electroporation. Induction of co-stimulatory molecule expression was entirely dependent on the TRIF pathway. Our results demonstrate that in human DCs the TRIF pathway is important for overall pro-inflammatory DC differentiation via TLR4 by mediating co-stimulation and playing a non-redundant role in pro-inflammatory cytokine induction.