AbstractGain- or loss-of-function mutations in fibroblast growth factor receptor 3 (FGFR3) result in cranial vault defects - highlighting the protein’s role in membranous ossification. Zebrafish express high levels offgfr3during skull development; in order to study FGFR3’s role in cranial vault development, we generated the firstfgfr3loss-of-function zebrafish(fgfr3lof/lof). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. Integrated analyses (in vivoimaging, and single-cell RNA sequencing of the osteoblast lineage) of zebrafishfgfr3lof/lofrevealed a delay in osteoblast expansion and differentiation, together with changes in the extracellular matrix. These findings demonstrate thatfgfr3is a positive regulator of osteogenesis. We hypothesize that changes in the extracellular matrix within growing bone impair cell-cell communication, mineralization, and new osteoblast recruitment.