PROJECT SUMMARY Heart failure is the deterioration of cardiac function in part due to cardiomyocyte (CM) death. In vitro differentiation of human pluripotent stem cells (hPSCs) into CMs is a key method for developing cell-replacement therapies for heart repair, due to the heart’s inability to regenerate. Despite improvements in differentiation efficiencies, current protocols give rise to functionally immature, diverse populations of CM sub-types at different developmental states leading to unfavorable effects when transplanted in humans. To generate specific, mature CM sub-types in vitro for heart repair treatment, it is essential to understand the cell fate decisions and developmental cues that allow a cell to become a particular cell type. A cell’s development is highly dependent on its environment within a tissue, particularly through cell-cell interactions between distinct cell types. To understand how CMs are influenced by their environment, we examined the differentiation states of CMs in a 2D (monolayer) versus 3D (embryoid body) cellular environment. Here, we observed that ventricular CMs (vCMs) develop more efficiently and CMs exhibit a more developmentally mature cellular state in the 3D environment as compared to the 2D environment. Additionally, the 3D system contains a more heterogenous cellular environment and has differing signaling cues versus the 2D system. Therefore, the central hypothesis of this proposal is that the non-cell autonomous effects of the surrounding 3D cellular environment may promote vCM differentiation by generating a more in vivo-like environment. Toward this end, we will investigate the impact of the 2D versus 3D cellular environments on vCM differentiation (Aim 1). Additionally, we will identify paracrine cues secreted from non-CM cell types that influence vCM differentiation (Aim 2). These key studies will elucidate the impact of non-CM cell types and their specific molecular cues on the generation of vCMs, thus allowing for the generation of pure populations of more in vivo-like cell types that can be used for heart failure treatment and therapeutic development.