Objective According to development biology, cranial neural crest stem cell (CNCSC) can differentiate into pre- cursor cells of tooth, jaw and peripheral nerve system, butin vitrostudy is less reported. In the present study, CNCSCwere dis- sociated and culturedin vitro,and the biological characteristics of CNCSCwas investigated.Methods Cranial neural tubes, dis- sected from mouse E9d, were explanted onto fibronectin-coated dishes. CNCSCs emigrated from the explanted neural tubes, cul- tured in a serum-free medium containing modified DMEM/F12.Biological characteristics of CNCSC were detected by morphology, nuclear labeled with BurdU and immunocytochemistry.Results Fibroblast-like CNCSCs demonstrated the characteristics of stem cell, such as clonality, self-renewal and multipotentiality. The result of immunocytochemical stain showed that CNCSC expressed HNK-1 antigen.Conclusion CNCSC were cultured successfully, providing a experimental basis for study on tooth/jaw-like dif- ferentiation of CNCSCin vitro, especially providing a cell source for investigating tooth/jaw regeneration.