Neurotrophic factors play an important role in the development and maintenance of not only neural but also nonneural tissues. Several neurotrophic factors are expressed in dental tissues, but their role in tooth development is not clear. Here, we report that neurotrophic factor neurotrophin (NT)-4 promotes differentiation of dental epithelial cells and enhances the expression of enamel matrix genes. Dental epithelial cells from 3-day-old mice expressed NT-4 and three variants of TrkB receptors for neurotrophins (full-length TrkB-FL and truncated TrkB-T1 and -T2). Dental epithelial cell line HAT-7 expressed these genes, similar to those in dental epithelial cells. We found that NT-4 reduced HAT-7 cell proliferation and induced the expression of enamel matrix genes, such as ameloblastin (Ambn). Transfection of HAT-7 cells with the TrkB-FL expression construct enhanced the NT-4-mediated induction of Ambn expression. This enhancement was blocked by K252a, an inhibitor for Trk tyrosine kinases. Phosphorylation of ERK1/2, a downstream molecule of TrkB, was induced in HAT-7 cells upon NT-4 treatment. TrkB-FL but not TrkB-T1 transfection increased the phosphorylation level of ERK1/2 in NT-4-treated HAT-7 cells. These results suggest that NT-4 induced Ambn expression via the TrkB-MAPK pathway. The p75 inhibitor TAT-pep5 decreased NT-4-mediated induction of the expression of Ambn, TrkB-FL, and TrkB-T1, suggesting that both high affinity and low affinity neurotrophin receptors were required for NT-4 activity. We found that NT-4-null mice developed a thin enamel layer and had a decrease in Ambn expression. Our results suggest that NT-4 regulates proliferation and differentiation of the dental epithelium and promotes production of the enamel matrix.