The lumen of the fallopian tube (FT) is lined with columnar epithelium composed of secretory and ciliated cells, both of which are important for reproduction. However, the molecular mechanism regulating cell fate remains controversial. In this study, we established a primary culture system using porcine fallopian tube epithelial cells (FTECs) to study the differentiation mechanism. We found that estrogen promoted the differentiation of multi-ciliated cells (MCCs) through estrogen receptor β, following the reduction of DLL1, a ligand of Notch. Meanwhile, epidermal growth factor (EGF), a regulator of epithelial homeostasis and differentiation, suppressed ciliogenesis by the activation of Notch signaling. However, the estrogen pathway did not affect the activation of the EGF pathway. Taken together, the differentiation of MMCs in FT depends on the balance of EGF and estrogen signaling, either of which inhibits or stimulates the Notch signaling pathway respectively.