Abstract: Current 2D in vitro monocultures and animal models fall short in accurately modeling cardiovascular genetic disorders, leading to inadequate therapeutic development. Recent advances have now centered on 3D organoid models due to their improved cellular heterogeneity. However, cardiac organoids still lack crucial structural characteristics, cell types, and cellular organization which limits their utility. While recent approaches focus on modifying morphogens, none have used mechanical cues as guides for the differentiation process. Preliminary data presented within reveal that microconfining stem cell colonies on compliant substrates facilitates cardiac organoid generation and release, with substrate stiffness and geometric asymmetry influencing the spatial organization of organoid structure. The research proposed in this fellowship will explore these findings further to develop a roadmap for engineering-in the missing structural components. By modulating matrix mechanics, we aim to replicate developmental stiffness, guiding traction stresses and cell polarization during early differentiation stages. Gradients and temporal changes of stiffness may push towards cardiac mesoderm specification via presentation of a more physiologically representative mechanical environment. Controlling local cell densities and geometric confinement may further play a role in guiding final organoid architecture and cell heterogeneity and distribution. Overall, the goal is this work is to establish a platform that can reproducibly create cardiac organoids with tunable spatial control for drug screening applications, with scope for application towards other organoid types.