The Nkx2-3 gene belongs to the NK2 class of homeobox genes that play important role in vertebrate development and share overlapping expression patterns. NK2s are involved in several pathways that lead to cell differentiation, migration, and maturation of the cells, indicating their essential role in the formation and homeostasis of the organism. Here we report that the Nkx2-3lacZ?HD/lacZ?HD mouse mutants have an orofacial phenotype affecting among other structures and the developing teeth. Specifically, we confirm that while incisors and upper molars are normal, the mandibular molars of the mutants have abnormal crown shape. Second, we provide for the first time evidence that the absence of Nkx2-3 affects the differentiation process of the two most essential dental cell populations, namely the ameloblasts and the odontoblasts. Macroscopic, histological, 3D micro-CT of the lower molars of Nkx2-3lacZDHD/lacZDHD mouse mutants exhibit enamel and dentin phenotypes with the expression of the Enamelin (Enam) and Dentin Sialophosphoprotein (DSPP) genes to be selectively reduced in Nkx2-3 deficient secretory ameloblasts and dentinoblasts, respectively.