Dysfunction of the cardiac pacemaker tissues due to genetic defects, acquired diseases, or aging results in arrhythmias. When arrhythmias occur, artificial pacemaker implants are used for treatment. However, the numerous limitations of electronic implants have prompted studies of biological pacemakers that can integrate into the myocardium providing a permanent cure. Embryonic stem (ES) cells cultured as three-dimensional (3D) spheroid aggregates termed embryoid bodies possess the ability to generate all cardiac myocyte subtypes. Here, we report the use of a SHOX2 promoter and a Cx30.2 enhancer to genetically identify and isolate ES cell-derived sinoatrial node (SAN) and atrioventricular node (AVN) cells, respectively. The ES cell-derived Shox2 and Cx30.2 cardiac myocytes exhibit a spider cell morphology and high intracellular calcium loading characteristic of pacemaker-nodal myocytes. These cells express abundant levels of pacemaker genes such as endogenous HCN4, Cx45, Cx30.2, Tbx2, and Tbx3. These cells were passaged, frozen, and thawed multiple times while maintaining their pacemaker-nodal phenotype. When cultured as 3D aggregates in an attempt to create a critical mass that simulates in vivo architecture, these cell lines exhibited an increase in the expression level of key regulators of cardiovascular development, such as GATA4 and GATA6 transcription factors. In addition, the aggregate culture system resulted in an increase in the expression level of several ion channels that play a major role in the spontaneous diastolic depolarization characteristic of pacemaker cells. We have isolated pure populations of SAN and AVN cells that will be useful tools for generating biological pacemakers.