Hydroxyapatite (HA) is the main constituent of bone mineral in vertebrates and is widely used as a biomaterial in biomedical studies and medical clinics. The synthesis of this calcium phosphate phase is a crucial factor in its potential use in various applications as well as in understanding the nucleation and growth processes in biological mineralization (biomineralization). In this work, a sequential analysis of the different steps of HA synthesis in aqueous media was performed by using cryo-transmission electron microscopy (Cryo-TEM), which allows preservation of the specimens at different stages of the synthesis process, preventing their modification during drying. X-ray energy dispersive spectroscopy and electron diffraction, in addition to high-resolution imaging, were performed on vitrified samples. We showed that drying a specimen from the synthesis solution induces the crystallization of nanoparticles into an HA phase after 30 min of reaction, while when analyzed by Cryo-TEM, crystalline nanoparticles were detected only after 15 h. Calcium phosphate nanoparticles presented three different morphologies: irregular shapes characterized by low contrast in TEM (5 min), isotropic shapes with well-defined contrast (10 min) and needle-like shapes (after 30 min). The long axis of the HA needle-like nanoparticles can be associated with the c-axis direction of the hexagonal unit cell, which can thus be assigned to the preferential crystallization direction. However, a diffraction signal of the nanoparticles in vitreous ice that “mimics” the original solution was detected by Cryo-TEM only after 15 h of synthesis. These apparently controversial results are discussed in this manuscript considering various aspects of the use of Cryo-TEM in relation to the current concepts of calcium phosphate nucleation and growth.