Objective To use a modified protocol to obtain in vitro-cultured mouse ependymal cells (MEPCs) and to assess the morphological change of MEPCs during differentiation, the process of cilia formation, and the ciliary motility of mature MEPCs. Methods MEPCs with physiological functions were obtained by specialized culture and induced differentiation from isolated neonatal mouse telencephalon cells. Cilia formation and maturation of MEPCs were confirmed by immunocytochemistry. The ciliary beating pattern was recorded using a microscope equipped with a high-speed camera. Results The isolated telencephalon cells proliferated rapidly under culture conditions, and MEPCs with functional multicilia were obtained through induced differentiation. Conclusions Using a modified in vitro culture protocol, MEPCs with proper immunohistochemical characteristics and normal multicilia motility were successfully obtained. The process of multicilia formation at various stages of MEPC differentiation was revealed, providing a benchmark for further study using MEPCs as a model.