It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligaseinactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligaseinactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKLinduced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.T NF receptor-associated factor 6 (TRAF6) is essential for many biological processes (1). These include the myeloid differentiation primary response gene 88 (MyD88) signaling network of the innate immune system, RANK ligand (RANKL)-dependent signaling and osteoclast formation, lymph node organogenesis (2), and the development of hair follicles, sweat glands, and sebaceous glands (3). TRAF6 expression is also needed for CD40 signaling in B cells (4), the maturation and development of dendritic cells (5), and the regulation of T-cell function (6, 7).In innate immunity, nearly all Toll-like receptors (TLRs), as well as the receptors of the interleukin 1 (IL-1) family of cytokines, initiate signaling by recruiting the adaptor protein MyD88. This is followed by the interaction of IL-1-receptor (IL-R)-associated kinase 4 (IRAK4) with MyD88 and then the interaction of other IRAK family members with IRAK4, to form an oligomeric complex, termed the Myddosome (8, 9). IRAK1 and IRAK2 can then interact with TRAF6 (10, 11) and induce TRAF6 dimerization (12), which triggers the activation of its E3 ligase activity (13).TRAF6 catalyzes the formation of Lys63-linked ubiquitin (K63-Ub) chains in vitro in the presence of Ubc13-Uev1a (also called UBE2N-UBE2V1), an E2 conjugating enzyme that directs the formation of this type of ubiquitin linkage (14, 15). Although truncated forms of TRAF6 lacking the really...