Biomineralization is a fundamental process key to the development of the skeleton. The phosphatase orphan phosphatase 1 (PHOSPHO1), which likely functions within extracellular matrix vesicles, has emerged as a critical regulator of biomineralization. The biochemical pathways which generate intravesicular PHOSPHO1 substrates are however currently unknown. We hypothesized that the enzyme ectonucleotide pyrophosphatase/phosphodiesterase (ENPP6) is an upstream source of PHOSPHO1 substrate.