Tooth development is initiated by epithelial-mesenchymal interactions via basement membrane (BM) and growth factors. In the present study, we found that nephronectin (Npnt), a component of the BM, is highly expressed in the developing tooth. Npnt localizes in the BM on the buccal side of the tooth germ and shows an expression pattern opposite that of the dental epithelial stem cell marker Sox2. To identify the roles of Npnt during tooth development, we performed knockdown and overexpression experiments using ex vivo organ and dental epithelial cell cultures. Our findings showed that loss of Npnt induced ectopic Sox2-positive cells and reduced tooth germ size. Over expression of Npnt showed increased proliferation, whereas the number of Sox2-positive cells was decreased in dental epithelial cells. Npnt contains 5 EGF-like repeat domains, as well as an RGD sequence and MAM domain. We found that the EGF-like repeats are critical for Sox2 expression and cell proliferation. Furthermore, Npnt activated the EGF receptor (EGFR) via the EGF-like repeat domains and induced the PI3K-Akt signaling pathway. Our results indicate that Npnt plays a critical scaffold role in dental epithelial stem cell differentiation and proliferation, and regulates Sox2 expression during tooth development.