Tooth development begins with the invagination of the epithelium and culminates in the formation of the mature tooth, a process regulated by various transcription factors. Pitt-Hopkins syndrome is a disorder characterized by craniofacial abnormalities; heterozygous mutations in the TCF4 gene are associated with distinctive facial features and severe psychomotor developmental delay. However, the role of TCF4 in tooth formation remains unclear. Using yeast two-hybrid and co-immunoprecipitation assays, we demonstrated that AmeloD, a tooth-specific transcription factor, binds directly to TCF4. Immunostaining and single-cell RNA sequencing analyses revealed that TCF4 is expressed in both dental epithelium and dental mesenchymal cells. Overexpression of AmeloD in the dental epithelial cell line SF2 induced TCF4 expression, whereas TCF4 overexpression did not induce AmeloD expression. Although stimulation with the nerve growth factor NT-4 normally induces the expression of differentiation markers such as Ambn and Enam in SF2 cells, we found that TCF4 overexpression inhibited this NT-4-induced cellular differentiation. Furthermore, this inhibitory effect was abolished by the overexpression of AmeloD. These findings reveal a novel function of TCF4, it directly inhibits AmeloD function through physical interaction. This insight is significant for elucidating the causes of the dental phenotypes observed in Pitt-Hopkins syndrome.