The crossed lamellar microstructure is the most commonly formed shell structure in mollusks. Although the mechanical properties and the architecture of the crossed lamellar microstructure have been extensively studied, its formation mechanisms are largely unknown. In this study, we obtained information on the formation processes of the aragonite crystals in the crossed lamellar microstructure using two converging approaches: cryo-scanning electron microscopy performed on the forming shell layer and in vitro crystallization experiments with extracted matrix macromolecules. We show that deposition of the first crystalline particles occurs in a thin granular layer on the growing surface of the mature crossed lamellar microstructure. The granules subsequently grow to form the lamellar prisms. In vitro, the ensemble of organic macromolecules extracted from the crossed lamellar layers induced the formation of only aragonite crystals from solutions containing Ca/Mg ion ratio of 1/2.5 within 6 h, and that the morphology and the crystallographic orientation of the synthetic crystals are the same as those of the biogenic crystals. In the absence of matrix macromolecules, both calcite and aragonite crystals formed. These observations provide insights into the early crystal nucleation and growth events of aragonite crystals in the formation of the crossed lamellar microstructure.