Using human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and beating embryoid bodies (EBs), we previously showed that intracellular Ca2+ -handling proteins developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological (EP) properties of hESC-CMs. Here, we verify our findings with mouse embryonic embryonic heart preparations at the organ level. We find that dominant pacemaking activity originated from the left inflow tract region at embryonic ages 8.5 days (E8.5), but switched to the right sinus node by E12.5. Additionally, we show that intracellular calcium-mediated automaticity develops early and is the major mechanism of pulse generation in the left inflow tract of E8.5 hearts. Later in development at E12.5, sarcolemmal ion channels develop in the sinus node at a time when pacemaker channels are down-regulated in the left inflow tract, leading to a switch in the dominant pacemaker location. Thus, we demonstrate that differential mechanistic development of automaticity between the left and right inflow tract regions confers the pacemaker location switch. Moreover, we find that a sodium channel independent sinus node- atrioventricular node (internodal) pathway mediates internodal conduction in E12.5 hearts.