Mouse ES cells can generate, through Embryoid Bodies (EBs) formation, sinoatrial-like myocytes. However these cells represent a small fraction of the whole EB. This work is aimed to isolate a homogeneous population of cardiac progenitors which specifically differentiate into pacemaker cardiomyocytes. We used the specific but transient expression of CD166 (a cell adhesion molecule) to isolate cardiac progenitors from differentiating EBs. CD166+ cells were sorted and analyzed by qRT-PCR, immunofluorescence and electrophysiology. The data show that CD166+ cells isolated from 8 days-old EBs express significantly higher levels of cardiac genes (sarcomeric a-actinin, Mef2c, cTnI, Gata4) than both CD166- and ES cells. CD166+ cells express transcription factors specifically involved in the generation of the conduction system (Shox2, Tbx18, Tbx3 and Isl-1) and genes typical of pacemaker myocytes (HCN4, HCN1, ssTnI) while expressing very low levels of ventricular genes (Nkx2-5, Cx43, HCN2). When CD166+ cells are plated, they form a layer of synchronously beating cells modulated by autonomic agonists. Importantly, these cells can be maintained in culture for more than four weeks without losing their pacemaker activity. In conclusion our data show that CD166+ cells isolated from differentiating EBs represent a population of pacemaker cardiomyocytes that can be cultured for long periods and can thus represent a suitable cellular pacemaker for cell therapy interventions.