Fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling has been linked to the aberrant mineralization phenotype of craniosynostosis syndromes. One critical aspect of mineralization involves the elaboration and transport of pyrophosphate into the extracellular matrix with subsequent enzymatic hydrolysis into phosphate. Altered expression of the pyrophosphate elaborating factors, TNAP (tissue nonspecific alkaline phosphatase), PC-1, and ANK, downstream of FGF/FGFR signaling may provide a potential mechanism for the craniosynostosis phenotype. As an initial step toward testing this hypothesis, we confirmed that ANK mRNA is upregulated during osteoblast differentiation in culture. Subsequently, the effect of FGF2 treatment on expression of PC-1, ANK, and TNAP in the calvarial osteoblastic cell line, MC3T3E1(C4), was investigated. FGF2 specifically induced expression of PC-1 and ANK while inhibiting expression of TNAP, at both mRNA and protein levels. Concordant with these changes in gene expression, FGF2 inhibited mineralization. These results suggest that FGF/FGFR signaling may affect mineralization via changes in the elaboration and metabolism of pyrophosphate.4641763184192