The cytokine interleukin-1 (IL-1) mediates immune and inflammatory responses by activating the transcription factor nuclear factor kappaB (NF-kappaB). Although transforming growth factor-beta-activated kinase 1 (TAK1) and mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3) are both crucial for IL-1-dependent activation of NF-kappaB, their potential functional and physical interactions remain unclear. Here, we showed that TAK1-mediated activation of NF-kappaB required the transient formation of a signaling complex that included tumor necrosis factor receptor-associated factor 6 (TRAF6), MEKK3, and TAK1. Site-specific, lysine 63-linked polyubiquitination of TAK1 at lysine 209, likely catalyzed by TRAF6 and Ubc13, was required for the formation of this complex. After TAK1-mediated activation of NF-kappaB, TRAF6 subsequently activated NF-kappaB through MEKK3 independently of TAK1, thereby establishing continuous activation of NF-kappaB, which was required for the production of sufficient cytokines. Therefore, we propose that the cooperative activation of NF-kappaB by two mechanistically and temporally distinct MEKK3-dependent pathways that diverge at TRAF6 critically contributes to immune and inflammatory systems.