The cell differentiation of the musculoskeletal system is highly coordinated during limb development. In the distal-most region of the limb, WNT and FGF released from the apical ectodermal ridge maintain mesenchymal cells in the undifferentiated stage. Once the cells stop receiving WNT and FGF, they respond to differentiation signals. Particularly during tendon development, mesenchymal cells enter the cell differentiation program once Scleraxis (Scx) gene expression occurs. Among the signals that trigger the cell differentiation programs, TGFβ signaling has been found to be closely involved in tendon differentiation. However, whether Scx gene expression depends merely on TGFβ signaling or other signals is still not fully understood. In the present study, considering that WNT/β-catenin is an inhibitory signal of cell differentiation, we speculated possible antagonistic or additive effects between canonical Wnt/β-catenin and TGFβ/SMAD signaling pathways to control Scx gene expression. We found that the blockade of WNT/β-catenin promoted Scx gene expression. In contrast, the inhibition of TGFβ/SMAD signaling did not maintain Scx gene expression. Interestingly, the blockade of both WNT/β-catenin and TGFβ/SMAD signaling at the same time promoted Scx gene expression. Thus the inhibition of WNT/β-catenin signaling appears to be necessary and sufficient to induce Scx gene expression.