Objective:To determine the differentiation capability of RAW 264.7 cells following subcultures. Methods:The cells were treated with receptor activator of NF-κB ligand (RANKL). The 6th,9th,13th,18th and 24th passages were treated with or without RANKL for 6 days. Tartrate-resistant acid phosphatase(TRAP) staining was used to observe the formation of osteoclasts. Results: RAW 264.7 cells at different passages all have TRAP-positive cells. There were no obvious differences among 6th,9th,13th and 18th in positive rate(P > 0.05),and the positive rate of 24th passage were significantly lower than that of other passages. Conclusion: RANKL can induce RAW 264.7 cells to form osteoclast. If cultured for long time, the differentiation capability of this cell line was reduced.