TGF-beta subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-beta signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-beta signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2(fl/fl);Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-beta2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-beta signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.