The aim of this study was to determine if embryonic stem cell derived cardiomyocyte aggregates (ESdCs) can act as pacemakers in spontaneously active cardiomyocyte preparations when their connexin isoform expression is tuned toward a more sinus nodal phenotype. Using microelectrode array recordings (MEAs), we demonstrate that mouse ESdCs establish electrical coupling with spontaneously active cardiomyocyte preparations (HL-1 monolayer) and obtain pacemaker dominance. WT- and Cx43(-/-)-ESdCs comparably established intercellular coupling with cardiac host tissue (Cx43(-/-): 86% vs. WT: 91%). Although both aggregates had a 100% success rate in pacing quiescent cardiac preparations, Cx43(-/-)-ESdCs had an increased likelihood of gaining pacemaker dominance (Cx43(-/-): 40% vs. WT: 13%) in spontaneously active preparations. No differences in size, beating frequency, V(m), or differentiation were detected between WT- and Cx43(-/-)-ESdCs but the intercellular coupling resistance in Cx43(-/-)-ESdCs was significantly increased (Cx43(-/-): 1.2nS vs. WT: 14.8nS). Lack of Cx43 prolonged the time until Cx43(-/-)-ESdCs established frequency synchronization with the host tissue. It further hampered the excitation spread from the cardiomyocyte preparation into the ESdC. However rectifying excitation spread in these co-cultures could not be unequivocally identified. In summary, ESdCs can function as dominant biological pacemakers and Cx43 expression is not a prerequisite for their electrical integration. Maintenance of pacemaker dominance depends critically on the pacemaker's gap junction expression benefiting those with increased intercellular coupling resistances. Our results provide important insight into the design of biological pacemakers that will benefit the use of cardiomyocytes for cell replacement therapy.