Ependymal cilia play pivotal roles in cerebrospinal fluid flow. In the primary culture system, undifferentiated glial cells differentiate well into ependymal multiciliated cells (MCCs) in the absence of fetal bovine serum (FBS). However, the substances included in FBS which inhibit this differentiation process have not been clarified yet. Here, we constructed the polarized primary culture system of ependymal cells using a permeable filter in which they retained ciliary movement. We found that transforming growth factor−1 (TGF−1) as well as Bone morphogenetic protein (BMP)−2 inhibited the differentiation with ciliary movement. The inhibition on the differentiation by FBS was recovered by the TGF−1 and BMP−2 inhibitors in combination.