Self-renewing embryonic stem (ES) cells have been established from early mouse embryos as permanent cell lines. By cultivation in vitro as three-dimensional aggregates called embryoid bodies (EBs), ES cells can differentiate into derivatives of all three primary germ layers, including cardiomyocytes. ES cells thus represent a useful model system for studying cardiomyocyte developmental paradigms. This chapter describes techniques and protocols for the cultivation and maintenance of ES cell lines, and the differentiation of ES cell lines into all specialized cell types of the heart, including atrial-, ventricular-, sinus nodal- and Purkinje-like cardiomyocytes. We also include protocols for the isolation and evaluation (morphological, molecular, and functional) of in vitro-generated cardiomyocytes. We consider these latter techniques to be prerequisites for the successful use of this model system to study cardiomyocyte differentiation. Finally, our objective in writing this chapter is to provide sufficient detail and explanation so that any competent scientist who is new to the field will be able to successfully establish and employ this model system for the analysis of ES cell-derived cardiomyocytes.