Micropatterned cocultures were fabricated with 3 extracellular matrices, hyaluronic acid (HA), fibronectin, and collagen. The feature of the fabrication processes is to avoid the use of potentially cytotoxic materials and utilize capillary force of the solution and interactions between the extracellular matrix components. The coculture system can be used to investigate the effects of heterocellular interactions on cellular fate. Direct heterocellular connections between hepatocytes and fibroblasts were visualized by the transcellular diffusion of fluorescein in this coculture system. The interactions between hepatocytes and fibroblasts were crucial for the maintenance of albumin synthesis by hepatocytes. The coculture system was also beneficial for investigating the effects of cell-cell interactions on the induction of embryonic stem (ES) cell differentiation. In cocultures grown in a sea-island pattern, ES cells formed isolated colonies surrounded by PA6 cells and differentiated into neurons with branched neurites that extended from the colonies. This versatile and biocompatible coculture system could potentially be a powerful tool for investigating cell-cell interaction and for tissue engineering applications.