TGF-β has been shown to inhibit and stimulate osteoclastogenesis. The purpose of this study was to evaluate the effects of TGF-β in hematopoietic cell cultures stimulated with RANKL and M-CSF. In cocultures of hematopoietic cells and BALC cells (a calvarial-derived cell line), TGF-β inhibited tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell formation. In contrast, TGF-β enhanced TRAP-positive multinucleated cell formation up to 10-fold in hematopoietic cell cultures containing few osteoblastic/stromal cells. Likewise, TGF-β increased the number of calcitonin receptor (CTR)-positive multinucleated and mononucleated cells in a concentration-dependent manner. An increase in cell size and multinuclearity was also observed in the presence of TGF-β. The stimulatory effects of TGF-β were dependent on the presence of M-CSF and RANKL. When differentiated on bovine cortical bone slices, these cells formed resorption lacunae. These results suggest that TGF-β has a direct stimulatory effect on osteoclastogenesis in hematopoietic cells treated with RANKL and M-CSF.