We present a computational fluid dynamics (CFD)-based model aimed at the identification of optimized culture conditions promoting efficient cardiogenesis of hydrogel-bead-encapsulated embryonic stem cells (ESCs) within a rotating bioreactor. The numerical approach, integrating diffusion, convection, and multiphase fluid dynamics calculations, allowed to evaluate (i) the microgravity motion of the floating beads, (ii) the O(2) delivery to the cells, also (iii) taking into account the cellular O(2) consumption, as a function of different rotation speeds of the breeding chamber. According to our results, a 25 rpm rotation (i) enhances an adequate mixing of the cell carriers, avoiding sedimentation and excessive packing, also maintaining a quite homogeneous distribution of the suspended beads and (ii) imparts a proper cellular O(2) supply, providing cells close to a normoxia condition. The bioreactor working conditions derived from the numerical analysis allowed the attainment of in vitro long-term cell viability maintenance, supporting efficient large-scale generation of ESC-derived cardiomyocytes (ESC-DCs) through a chemical-based conditioning bioprocess. In conclusion, we demonstrated the feasibility of using CFD-based tools, as a reliable and cost-effective strategy to assist the design of a 3D cardiogenic bioprocess.