AbstractCranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. CrossingAxin2^LacZ/+mouse, endowing enhanced Wnt activation, to aTwist1^+/−mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy inTwist1^+/−mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis.