We have explored the role of Wnt signaling in dentinogenesis of mouse molar teeth. We found that Wnt10a was specifically associated with the differentiation of odontoblasts and that it showed striking colocalization with dentin sialophosphoprotein (Dspp) expression in secretory odontoblasts. Dspp is a tooth specific non-collagenous matrix protein and regulates dentin mineralization. Transient overexpression of Wnt10 in C3H10T1/2, a pluripotent fibroblast cell line induced Dspp mRNA. Interestingly, this induction occurred only when transfected cells were cultured on Matrigel basement membrane extracts. These findings indicated that Wnt10a is an upstream regulatory molecule for Dspp expression, and that cell-matrix interaction is essential for induction of Dspp expression. Furthermore, Wnt10a was specifically expressed in the epithelial signaling centers regulating tooth development, the primary and secondary enamel knots. The spatial and temporal distribution of Wnt10a mRNA demonstrated that the expression shifts from the secondary enamel knots, to the underlying preodontoblasts in the tips of future cusps. The expression patterns and overexpression studies together indicate that Wnt10a is a key molecule for dentinogenesis and that it is associated with the cell-matrix interactions regulating odontoblast differentiation. We conclude that Wnt10a may link the differentiation of odontoblasts and cusp morphogenesis.