Conditionally immortalized murine calvarial (CIMC) cells that support differentiation of precursors into mature osteoclasts were isolated. All six CIMC cell lines support osteoclast differentiation in response to 1,25-dihydroxyvitamin D₃ or IL-11. CIMC-4 cells also support osteoclast differentiation in response to TNF, IL-1ß, or IL-6. The resultant multinucleated cells express tartrate-resistant acid phosphatase and form 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 stimulate differentiation when osteoclast precursors are co-cultured with either CIMC-4 cells or primary calvarial cells. Osteoclast differentiation induced by all stimuli other than TNF is completely blocked by osteoprotegerin, whether the stimulators are examined alone or in combination. Moreover, 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.