Effects of various colony‐stimulating factors (CSFs) [interleukin‐3 (IL‐3), granulocyte‐macrophage CSF (GM‐CSF), macrophage CSF (M‐CSF), and granulocyte CSF (G‐CSF)] on osteoclast‐like cell formation were examined in two different culture systems: the one‐step mouse marrow culture system and the two‐step coculture system of mouse primary osteoblastic cells with the bone marrow cells collected from the colonies that formed in the methylcellulose in the presence of the CSFs. In the one‐step mouse marrow cultures, none of the CSFs stimulated the formation of tartrate‐resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts)‐positive multinucleated cells (MNCs). Furthermore, the CSFs other than G‐CSF inhibited in a dose‐dependent manner the TRAP‐positive MNC formation induced by 1α‐25‐dihydroxyvitamin D₃ [1α,25‐(OH)₂D₃]. In contrast, when marrow cells were first cultured in semisolid methylcellulose in the presence of a CSF and the recovered marrow cells from the semisolid cultures were subsequently cocultured with primary osteoblastic cells in the presence of 1α,25‐(OH)₂D₃, numerous TRAP‐positive MNCs were formed. [¹²⁵I]salmon calcitonin specifically bound to TRAP‐positive cells formed in this two‐step culture system. Over 90% of the TRAP‐positive mononuclear cells and MNCs accumulated [¹²⁵I]calcitonin. M‐CSF was the most potent in inducing TRAP‐positive MNCs, followed by GM‐CSF, IL‐3, and G‐CSF in that order. No TRAP‐positive cells were formed in the absence of either osteoblastic cells or 1α,25‐(OH)₂D₃. These results clearly indicate that the CSFs, especially M‐CSF, stimulate the growth of osteoclast progenitors, which differentiate into osteoclast‐like cells in response to 1α,25‐(OH)₂D₃ in the presence of osteoblastic cells.