To delineate the competence window in which canonical wingless (Wnt)-signaling can either inhibit or promote osteogenic differentiation, we have analyzed cells with different status, specifically undifferentiated mesenchymal cells, such as adipose-derived stem cells and embryonic calvarial mesenchymal cells, and differentiated mesenchymal cells such as juvenile immature calvarial osteoblasts and adult calvarial osteoblasts. Our analysis indicated that undifferentiated mesenchymal cells and juvenile calvarial osteoblasts are endowed with higher levels of endogenous canonical Wnt signaling compared to fully differentiated adult calvarial osteoblasts, and that different levels of activation inversely correlated with expression levels of several Wnt antagonists. We have observed that activation of canonical Wnt signaling may elicit opposite biological activity in the context of osteogenic differentiation depending on the status of cell, the threshold levels of its activation, and Wnt ligands concentration. The results presented in this study indicate that treatment with Wnt3 and/or expression of constitutively activated β-catenin inhibits osteogenic differentiation of undifferentiated mesenchymal cells, whereas expression of dominant negative transcription factor 4 (Tcf4) and/or secreted frizzled related protein 1 treatment enhances their osteogenic differentiation. Wnt3a treatment also inhibits osteogenesis in juvenile calvarial osteoblasts in a dose-dependent fashion. Conversely, Wnt3a treatment strongly induces osteogenesis in mature calvarial osteoblasts in a dose-dependent manner. Importantly, in vitro data correlated with in vivo results showing that Wnt3a treatment of calvarial defects, created in juvenile mice, promotes calvarial healing and bone regeneration only at low doses, whereas high doses of Wnt3a impairs tissue regeneration. In contrast, high doses of Wnt3a enhance bony tissue regeneration and calvarial healing in adult mice. Therefore, the knowledge of both endogenous activity of canonical Wnt signaling and appropriate concentrations of Wnt3a treatment may lead to significant improvement for bony tissue engineering, as well as for the efficient implement of adipose-derived stem cells in bone regeneration. Indeed, this study has important potential implications for tissue engineering, specifically for repair of juvenile bone defects.