Activation of IκB kinase (IKK) is the key step in stimulation of the transcription factor NF-κB, which regulates many genes in the inflammatory response pathway. The molecular mechanism that underlies IKK activation in response to tumor necrosis factor (TNF) is still unknown. Using mitogen-activated protein kinase kinase kinase 3 (MEKK3)-deficient fibroblast cells, we found that MEKK3 plays a critical role in TNF-induced NF-κB activation. We have shown that MEKK3 is required for IKK activation and functions downstream of receptor-interacting protein (RIP) and TNF receptor–associated factor 2. We have also shown that MEKK3 interacts with RIP and directly phosphorylates IKK. The kinase activity of MEKK3 is pivotal to its function and, therefore, MEKK3 links RIP and IKK in TNF-induced NF-κB activation.