Abstract: The purpose of this study was to investigate an effective culture method of osteoclasts(OC) formation and to test the possibility that chODF (chicken osteoclast differentiation factor) was sufficient for OC development in primary chicken embryo marrow cell culture. Femurs and humerus were dissected and bone marrow cells were collected and cultured in seven culture methods according to the medium with different concentration of chODF, M-CSF and chOPG. OC formation was observed and indentified by TRAP (tartrateresistant acid phosphatase), HE, toluidine blue staining, phasecontrast microscope and scanning electron microscopy. Results showed that the combination of chODF and M-CSF could induce OLCs formation and bone resorption in chicken embryo marrow cell culture. The number of TRAPpositive multi-nucleated cells in the culture medium increased dosedependently with ODF concentration (P<0.05), at meanwhile, OPG can interrupt the formation of OLC. In conclusion, we established an effective way to form OLC, and it could be employed to investigate the differentiation and activation of OC. It also provide theory of mechanism of OPG/ODF/RANK in the process of osteoporosis in layer hens.