TRIF is an essential adaptor for Toll-like receptor 3/4 (TLR3/4) signaling to activate transcription factor interferon regulatory factor 3 (IRF-3). We examined the molecular mechanism of TLR3 signaling and found that TLR3 stimulation by double-stranded RNA (dsRNA) induces phosphorylation of TRIF at Ser210 and is required for IRF-3 recruitment. TANK-binding kinase 1 (TBK1) is known to be responsible for IRF-3 phosphorylation and activation. We found that TBK1 is also responsible for phosphorylation of Ser210 in TRIF. Unexpectedly, we discovered that IB kinase β (IKKβ) plays an essential role in TLR3/4 signaling using a pharmacological inhibitor and gene deletion. Of note, IKKβ is essential in TLR3/4 but not in retinoic acid-inducible gene I (RIG-I) signaling. Mechanistically, IKKβ transiently associates with and induces the phosphorylation of TBK1 upon TLR3 stimulation. These results suggest a phosphorylation cascade of IKKβ and TBK1, where priming phosphorylation of TBK1 by IKKβ is required to surpass the threshold to induce signaling, thereby activating IRF-3.