Phosphate and parathyroid hormone related peptide (PTHrP) are required for normal growth plate maturation. Hypophosphatemia impairs hypertrophic chondrocyte apoptosis leading to rachitic expansion of the growth plate; however, the effect of phosphate restriction on chondrocyte differentiation during endochondral bone formation has not been examined. Investigations were, therefore, undertaken to address whether phosphate restriction alters the maturation of embryonic d15.5 murine metatarsal elements. Metatarsals cultured in low phosphate media exhibited impaired chondrocyte differentiation, analogous to that seen with PTHrP-treatment of metatarsals cultured in control media. Because phosphate restriction acutely increases PTHrP expression in cultured metatarsals, studies were undertaken to determine if this increase in PTHrP plays a pathogenic role in the impaired chondrocyte differentiation observed under low phosphate conditions. In contrast to what was observed with wild-type metatarsal elements, phosphate restriction did not impair the differentiation of metatarsals isolated from PTHrP heterozygous or PTHrP knockout mice. In vivo studies in postnatal mice demonstrated that PTHrP haploinsufficiency also prevents the impaired hypertrophic chondrocyte apoptosis observed with phosphate restriction. To determine how signaling through the PTH/PTHrP receptor antagonizes the pro-apoptotic effects of phosphate, investigations were performed in primary murine hypertrophic chondrocytes. Receptor activation impaired phosphate-induced Erk1/2 phosphorylation specifically in the mitochondrial fraction and decreased levels of mitochondrial Bad, while increasing cytosolic phospho-Bad. Thus, these data demonstrate that phosphate restriction attenuates chondrocyte differentiation as well as impairing hypertrophic chondrocyte apoptosis and implicate a functional role for the PTH/PTHrP signaling pathway in the abnormalities in chondrocyte differentiation and hypertrophic chondrocyte apoptosis observed under phosphate restricted conditions.