ABSTRACT The calvaria (upper part of the skull) comprises plates of bone and fibrous joints (sutures and fontanels). While the bone protects the brain, the sutures contain stem cells for osteoblasts, and thus allow the skull to grow coordinately with the expanding brain of a child. Craniosynostosis (a premature loss of the suture(s)) is a major class of human birth defects. It can lead to dysmorphic skull, and further affect brain and orofacial development. Current treatment for craniosynostosis often involves invasive and repetitive surgeries at young ages with relatively high rates of complications. Therefore, improving the methods of intervention for this defect is of great importance to public health. The calvaria is made of cells from the neural crest and the mesoderm in embryos. In mice, these cells form a mesenchyme layer that completely encases the brain soon after mid-gestation (cranial mesenchyme). The cranial mesenchyme on the apical side of the head (a.k.a. early migrating mesenchyme, EMM) gives rise to soft tissues such as the sutures, the dermis/hypodermis of the scalp, and the meninges. Our previous study has shown that LMX1B (LIM homeobox transcription factor 1b) plays a key role in suppressing osteogenesis in EMM to allow suture formation. Recently, single cell sequencing and in silico analyses from our group and others have suggested that EMM contains two populations of intermediate progenitors: suturodermal progenitors (SDP) with a dual potential for the suture mesenchyme and the dermis/hypodermis, and common meningeal progenitors (CMP), which contribute to the dura and the arachnoid layers of the meninges. We found that both SDP and CMP populations were severely decreased in Lmx1b mutants, whereas cells with a restricted fate were increased and appeared precociously. The goal of this project is to identify factors underpinning cell fate specification and differentiation in the cranial mesenchyme, and to utilize this information to generate SDP in vitro from human embryonic stem cells (hESC). In Aim1. we will determine transcription factors and signaling pathways that regulate cell fate specification and differentiation in the cranial mesenchyme in vivo. In Aim2, we will determine genetic regulators that control SDP specification and differentiation in vitro from hESC. The completion of this project will fill the critical gap in the current knowledge of suturogenesis. Furthermore, our results can facilitate efforts to use suture stem cells for craniosynostosis treatment, a promising new direction of research in the field.