The Musashi family of RNA-binding proteins, Musashi1 and Musashi2, regulate self-renewal and differentiation of neuronal and hematopoietic stem cells by modulating protein translation. It has been recently reported that Musashi2, not Musashi1, regulates hematopoietic stem cells. Although osteoclasts are derived from hematopoietic cells, the expression and functions of Musashi proteins in osteoclast lineage cells remain unknown. In this study, we have uncovered that Musashi2 is the predominant isoform of Musashi proteins in osteoclast precursors and its expression is upregulated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. Knocking down the expression of Musashi2 in osteoclast lineage cells by shRNAs attenuates nuclear factor of activated T cells 1 (NFATc1) expression and osteoclast formation in vitro. Mechanistically, loss of Musashi2 inhibits Notch signaling during osteoclast differentiation and induces apoptosis in pre-osteoclasts. In contrast, depletion of Musashi2 has no effects on cell cycle progression and p21(WAF-1) protein expression in macrophages. Furthermore, depletion of Notch2 and its downstream target Hes1 in osteoclast precursors by shRNAs abrogates osteoclastogenesis by inhibiting NFATc1. Finally, absence of Musashi2 in osteoclast precursors promotes apoptosis and inhibits RANKL-induced nuclear factor-κB (NF-κB) activation, which is essential for osteoclast survival, Thus, Musashi2 is required for cell survival and optimal osteoclastogenesis by affecting Notch signaling and NF-κB activation.