Virus infection, double-stranded RNA, and lipopolysaccharide each induce the expression of genes encoding IFN-α and -β and chemokines, such as RANTES (regulated on activation, normal T cell expressed and secreted) and IP-10 (IFN-γ inducible protein 10). This induction requires the coordinate activation of several transcription factors, including IFN-regulatory factor 3 (IRF3). The signaling pathways leading to IRF3 activation are triggered by the binding of pathogen-specific products to Toll-like receptors and culminate in the phosphorylation of specific serine residues in the C terminus of IRF3. Recent studies of human cell lines in culture have implicated two noncanonical IκB kinase (IKK)-related kinases, IKK-ε and Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1), in the phosphorylation of IRF3. Here, we show that purified recombinant IKK-ε and TBK1 directly phosphorylate the critical serine residues in IRF3. We have also examined the expression of IRF3-dependent genes in mouse embryonic fibroblasts (MEFs) derived from Tbk1^-/- mice, and we show that TBK1 is required for the activation and nuclear translocation of IRF3 in these cells. Moreover, Tbk1^-/- MEFs show marked defects in IFN-α and -β, IP-10, and RANTES gene expression after infection with either Sendai or Newcastle disease viruses or after engagement of the Toll-like receptors 3 and 4 by double-stranded RNA and lipopolysaccharide, respectively. Finally, TRIF (TIR domain-containing adapter-inducing IFN-β), fails to activate IRF3-dependent genes in Tbk1^-/- MEFs. We conclude that TBK1 is essential for IRF3-dependent antiviral gene expression.