Currently, only limited insight into mechanisms promoting the differentiation and specification of the mammalian cardiac conduction system is available. Therefore, we established a murine embryonic stem (ES) cell line stably expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of the human atrial natriuretic peptide (ANP) promoter to further characterize the development of very early stages of the mammalian cardiac conduction tissue. The cardiac nature of ANP-EGFP positive cells was confirmed by immunostaining. In ANP-EGFP expressing ES cell-derived cardiomyocytes, a distinct sublineage of pacemaker cells could be identified. Pacemaker cells displayed a spindle shape and exhibited a higher spontaneous beating rate, faster If current activation and larger If current densities compared with triangular atrial-like cardiocytes. Exposure to endothelin-1 significantly increased the percentage of pacemaker-like cells without affecting their electrophysiological properties. These findings were corroborated by immunostaining with antibodies against connexin 40 and connexin 45, known markers for cardiac conduction tissue. Conversely, treatment of ANP-EGFP expressing ES cells with neuregulin-1 exhibited no effect on differentiation. These results indicate that ANP-EGFP expression enables the identification of ES cell-derived pacemaker cells by their fluorescence and morphology and that endothelin-1 promotes the development of ANP-EGFP positive cardiomyocytes to a pacemaker-like phenotype.