Background/Aim: The mouse vagina exhibits stratified squamous epithelium, which is comprised of multiple cell layers. We previously showed that erbB signaling, induced by epithelial estrogen receptor 1 (ESR1), is required for the initial differentiation of the epithelium. However, the downstream effector that mediates terminal differentiation in the apical layers remains elusive. The contribution of fibroblast growth factor (FGF) to vaginal epithelial cell differentiation was investigated. Materials and Methods: Vaginas from wild-type or epithelium-specific Esr1 conditional knockout (cKO) mice were analyzed using immunohistochemistry and quantitative real-time RT-PCR. Results: Of the FGF ligands examined, Fgf22 mRNA was significantly induced following estrogen treatment. Furthermore, FGF downstream signaling, phosphorylated FRS2 and ERK1/2 were exclusively expressed in the apical layers of the vaginal epithelium. No changes in such expression were observed in the Esr1 cKO mice. Conclusion: FGF-ERK/MAPK pathway may be a main inducer of terminal differentiation in the mouse vaginal epithelium.