When mouse bone marrow cells were co-cultured with a stromal cell line. ST2, numerous osteoclast-like multinucleated cells (MNCs) were formed. To enrich the MNCs which were tartrate-resistant acid phosphatase (TRAP)-positive, we treated the cultures with dispase. Enriched TRAP-positive MNCs adhering to the bottom of the dish were cultured for a further 24h with medium containing 15% fetal calf serum. More than 80% of TRAP-positive MNCs were detached from the dish during the culture. However, when ST2 cells were included during the culture period, survival of TRAP-positive MNCs were significantly increased. Moreover, among various factors examined for the survival of TRAP-positive MNCs, only M-CSF and IL-1 alpha were effective in prolonging viability of TRAP-positive MNCs.