BACKGROUND: The differentiation of pluripotent stem cells (PSCs) and their self-organization into organoids are influenced by cell-cell interactions mediated by contacts and secreted molecules. These interactions are enhanced in microfluidic droplets due to confinement and small culture volumes. However, a comprehensive study on the culture of PSCs within droplets and the impact of this microenvironment has yet to be conducted. METHODS: In this study, we present a droplet platform for the 3D culture of PSCs at various stages of cellular commitment, such as pluripotency, spontaneous differentiation and directed differentiation towards gastruloids and cardioids. RESULTS: We demonstrate PSC differentiation into the three germ layers and the feasibility of organoid formation within droplets. Our findings reveal that culturing PSCs in confined volumes regulates cell fate decisions, promoting tissue patterning in gastruloids through the sequential induction of growth and migration of distinct differentiated cell populations, and facilitating the self-organization of cardiac organoids. CONCLUSION: This technological approach provides unique insights into the intrinsic factors regulating tissue self-patterning in vitro, with potential applications in drug screening and disease modeling.