Embryonic stem cells (ESCs) are promising cell sources for cell-based therapy. It has been established that the formation of ESC aggregates promotes their differentiation into the derivatives of all three germ layers. ESC aggregates are generally prepared via the formation of suspended spherical aggregates called embryoid bodies (EBs). Because the differentiation efficiency depends on the size of EBs, it becomes one of the research topics how to prepare size-controlled EBs in a scalable manner for reproducible and high-throughput experiments. Here, we have developed a novel culture method that enables simple mass preparation of size-controlled ESC aggregates on a culture surface instead of floating EBs. We developed a maskless photolithography device that enabled rapid fabrication of micropatterned surfaces. Utilizing this device, we fabricated the culture substrates the surfaces of which comprised arrays of cell-adhesive circular micro-domains (100-400 microm in diameter) and the rest of non-cell-adhesive domains. We seeded mouse ESCs on this substrate and prepared size-controlled ESC aggregates on the micro-domains. We analyzed cardiac differentiation in the ESC aggregates and found that the optimal diameter of micro-domains was 200 microm. The present method is useful for the simple and reproducible mass preparation of ESC-derived differentiated cells and high-throughput assays.