Objective: This study aimed to establish and characterize ectomesenchymal stem cells and further reveal the effects of all trans-retinoic acid (RA) on the biologic behaviors of these cells. Methods: Ectomesenchymal stem (EMS) cells of developing palatal processes were explanted from the palatal shelves of embryonic BALB/c mice. The characterization of EMS cells was accomplished by immunohistochemical analysis and growth curves. The action of RA and EMS cells was evaluated according to population doubling time and the terminal deoxyribonucleotidyl transferase dUTP nick end-labeling assay. Results: Primary culturing of EMS cells proceeded successfully. The results of immunohistochemistry showed that EMS cells proceeded successfully. The results of immunohistochemistry showed that EMS cells stained neuron-specific enolase (+), S-100 (+), vimentin (+++), keratin (-), myoglobin (-) and factor VIII (-). The population doubling time of RA-treated EMS cells was much longer compared with nontreated EMS cells. RA also dramatically increased the number of apoptotic cells. Conclusion: Ectomesenchymal stem cells may be undifferentiated. RA inhibited their proliferation and induced apoptosis. The inhibition of growth and excess apoptosis may contribute to the formation of cleft lip/palate and other orofacial congenital malformations.