Embryonic stem (ES) cells hold great potential as a renewable cell source for regenerative medicine and cell-based therapy. Despite the potential of ES cells, conventional stem cell culture methods do not enable the control of the microenvironment. A number of microscale engineering approaches have been recently developed to control the extracellular microenvironment and to direct embryonic stem cell fate. Here, we used engineered concave microwell arrays to regulate the size and shape of embryoid bodies (EBs)-cell aggregate intermediates derived from ES cells. Murine ES cells were aggregated within concave microwells, and their aggregate sizes were controlled by varying the microwell widths (200, 500, and 1000 mum). Differentiation of murine ES cells into three germ layers was assessed by analyzing gene expression. We found that ES cell-derived cardiogenesis and neurogenesis were strongly regulated by the EB size, showing that larger concave microwell arrays induced more neuronal and cardiomyocyte differentiation than did smaller microwell arrays. Therefore, this engineered concave microwell array could be a potentially useful tool for controlling ES cell behavior.