AbstractDendritic cell activation by Toll‐like receptors (TLR) is crucial for the generation of protective immune responses. In addition to the common myeloid differentiation factor 88 (MyD88)‐dependent signaling pathway, TLR4 engages the adaptor protein Toll/IL‐1 receptor (TIR)‐domain‐containing adaptor inducing IFN‐β (TRIF), leading to interferon regulatory factor 3 (IRF‐3) activation and type I interferon production. Using microarray expression profiling we now identify TRIF as a major regulator of the TLR4‐triggered activation program of dendritic cells. We show that the expressionof 47% of the genes that are responsive to TLR4 stimulation in wild‐type dendritic cells is significantly altered in cells carrying a loss‐of‐function mutation of TRIF. Specifically, expression of IL‐12, IL‐18, and IL‐23 was impaired in the absence of functional TRIF, suggesting that TLR4‐promoted Th1 responses are TRIF‐dependent. Furthermore, we provide evidence that TRIF regulates TLR4‐mediated gene expression both by type I IFN‐dependent and ‐independent mechanisms. Whereas dendritic cell production of CXCL10 and CCL12 was dependent on both TRIF and the type I interferon receptor, expression of IL‐6 required TRIF but not type I interferon activity. Functional TRIF was also required for the normal induction of numerous genes considered important for host defense against diverse pathogens.Together, these data therefore identify TRIF as a crucial regulator of TLR4‐dependent dendritic cell responses.