Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. Recent evidence indicates that the TNF-related apoptosis-inducing ligand (TRAIL) of the TNF ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated T cells. We show in this work that TRAIL can induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. We demonstrated that both cell models differentiate into osteoclast-like cells in the presence of TRAIL in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. The TRAIL-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. However, TRAIL-induced osteoclastogenesis is dependent on activation of NF-£eB, ERK, and p38 MAP kinase. The TRAIL-induced osteoclastogenesis was significantly inhibited by treatment with TRAF-6 siRNA and TRAF-6 decoy peptide, indicating this pathway is TRAF-6 dependent. Thus, our data demonstrate that TRAIL induces osteoclast differentiation via direct engagement with the TRAIL death receptor through a signaling pathway distinct from apoptosis. Our results indicate that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation. It provides a novel role for TRAIL in regulating osteoclast differentiation and in osteoimmunology.