Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/β-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor lead to human tooth agenesis. We have investigated the functions of Wnt/β-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/β-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/β-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/β-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. , a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors and in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that / antagonistic interactions modulate the level of mesenchymal Wnt/β-catenin signaling, underlying the contrasting dental phenotypes caused by human and mutations.