Pure populations of cardiomyocytes derived from embryonic stem cells offer great promise as potential cell replacement therapies, as well as for use in pharmaceutical studies. The differentiation process, however, is frequently inefficient and nonspecific. We sought to improve upon the current processes by coupling automation and Design of Experiment (DOE). DOE-generated combinations of pro-cardiomyocyte compounds were converted to dispensing volumes using Automated Assay Optimization (AAO) for BioRAPTR* software, and reagents and cells were dispensed into 384-well plates using the BioRAPTR FRD* Microfluidic Workstation. After five days of culture, the resulting embryoid bodies (EBs) were transferred to 96-well gelatin-coated plates using the Biomek FXP* liquid handler. Over an additional three days, the wells were observed for spontaneously beating regions of cardiomyocytes, and the differentiated cells were further analyzed using flow cytometry to detect myosin heavy chain as a marker of cardiomyocytes. This bulletin illustrates the potential of a system that can both automate and optimize the differentiation of murine embryonic stem cells.