Conditionally immortalized murine calvarial (CIMC) cells that support differentiation of precursors into mature osteoclasts were isolated. All six CIMC cell lines supported osteoclast differentiation in response to 1,25-dihydroxyvitamin D₃ or interleukin (IL)-11. CIMC-4 cells also supported osteoclast differentiation in response to tumor necrosis factor (TNF)-, IL-1, or IL-6. The resultant multinucleated cells expressed tartrate-resistant acid phosphatase and formed resorption lacunae on mineralized surfaces. CIMC-4 cells, therefore, establish an osteoclast differentiation assay that is responsive to many cytokines and does not rely on isolation of primary stromal support cells. Low concentrations of the cytokines synergistically stimulated differentiation when osteoclast precursors were cocultured with either CIMC-4 cells or primary calvarial cells. Osteoclast differentiation induced by all stimuli other than TNF- was completely blocked by osteoprotegerin, whether the stimulators were examined alone or in combination. Moreover, study of precursors that lack TNF- receptors showed that TNF- induces osteoclast differentiation primarily through direct actions on osteoclast precursors, which is a distinct mechanism from that used by the other bone-resorptive agents examined in this study.