Abstract The primary objective of this application is to elucidate the regulatory mechanisms underlying odontogenesis mediated by nonclassical β-catenin signaling. Tooth agenesis is the most common congenital dental abnormality, characterized by the absence of one or more permanent teeth including Anodontia, Oligodontia, and Hypodontia. Human genetic studies of nonsyndromic tooth agenesis have revealed approximately 16 causative genes of which 6 of them are involved in the Wnt pathway indicating its significance in disease pathogenesis. Missing teeth are currently treated by dental implants, tooth transplants, or prosthodontic repairs. However, they are not permanent treatments and also are associated with considerable complications. For next-generation therapies, new regenerative approaches to interrupt tooth formation/maintenance or develop an autologous tooth replacement are highly attractive concepts. Therefore, it is critical to advance our knowledge of odontogenesis and elucidate the mechanisms underlying reciprocal interactions of dental epithelium and mesenchyme. Canonical Wnt signaling mediated by β-catenin has been well established to play an essential role in early odontogenesis. Mouse genetic studies have demonstrated the importance of Wnt signaling in various aspects of dental medicine, e.g. tooth development, dental pulp, enamel, odontogenesis, and amelogenesis. Expression of a degradation-deficient form of β-catenin causes continuous tooth generation and development of supernumerary teeth, further suggesting that tooth renewal can be unlocked by increasing the intrinsic level of odontogenic potential. β-catenin acts as a master regulator of this intrinsic potential to promote tooth formation. However, β-catenin is a multifaced protein that possesses other functions in addition to acting as a master regulator for transducing canonical Wnt signaling. Preliminary studies of our new genetic models argue against current knowledge and implicate the requirement of nonclassical β-catenin in odontogenesis. First, we will characterize new β-catenin mutant mice to rigorously assess the dual function of β-catenin in the development of odontogenic ability, and differentiation of odontoblasts and ameloblasts. Second, we will identify the master regulators acting downstream of β-catenin by a single-cell transcriptomic approach to decipher the genetic regulatory network associated with the nonclassical signaling cascade. The objective of this study has great significance in human health and regenerative dental medicine. Elucidating the mechanism underlying the regulation of odontogenesis promises important insights into next-generation therapy for dental restoration.