Bone mineralization is initiated by matrix vesicles, small extracellular vesicles secreted by osteoblasts, inducing the nucleation and subsequent growth of calcium phosphate crystals inside. Although calcium ions (Ca2+) are abundant throughout the tissue fluid close to the matrix vesicles, the influx of phosphate ions (PO43−) into matrix vesicles is a critical process mediated by several enzymes and transporters such as ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis (ANK), and tissue nonspecific alkaline phosphatase (TNSALP). The catalytic activity of ENPP1 in osteoblasts generates inorganic pyrophosphate (PPi) intracellularly and extracellularly, and ANK may allow the intracellular PPi to pass through the plasma membrane to the outside of the osteoblasts. Although the extracellular PPi binds to growing hydroxyapatite crystals to prevent crystal overgrowth, TNSALP on the osteoblasts and matrix vesicles hydrolyzes PPi into PO43− monomers: the prevention of crystal growth is blocked, and PO43− monomers are supplied to matrix vesicles. In addition, PHOSPHO1 is thought to function inside matrix vesicles to catalyze phosphocoline, a constituent of the plasma membrane, consequently increasing PO43− in the vesicles. Accumulation of Ca2+ and PO43− inside the matrix vesicles then initiates crystalline nucleation associated with the inner leaflet of the matrix vesicles. Calcium phosphate crystals elongate radially, penetrate the matrix vesicle’s membrane, and finally grow out of the vesicles to form calcifying nodules, globular assemblies of needle-shaped mineral crystals retaining some of those transporters and enzymes. The subsequent growth of calcifying nodules appears to be regulated by surrounding organic compounds, finally leading to collagen mineralization.