Teeth consist of two major tissues, enamel and dentin, which are formed during development by epithelial and mesenchymal cells, respectively. Rodent incisors are useful experimental models for studying the molecular mechanisms of tooth formation because they are simultaneously growing in not only embryos but also adults. Members of the transforming growth factor-β (TGF-β) family regulate epithelial-mesenchymal interactions through an essential coactivator, Smad4. In the present study, we established Smad4 conditional knockout (cKO) mice and examined phenotypes in adult incisors. Smad4 cKO mice died with severe anemia within one month. Phosphorylated Smad1/5/9 and Smad2/3 were detected in epithelial cells in both control and Smad4 cKO mice. Disorganized and hypoplastic epithelial cells, such as ameloblasts, were observed in Smad4 cKO mice. Moreover, alkaline phosphatase expression and iron accumulation were reduced in dental epithelial cells in Smad4 cKO mice. These findings suggest that TGF-β family signaling through Smad4 is required for the differentiation and functions of dental epithelial cells in adult mouse incisors.