Our recent studies have identified a Shox2-Nkx2-5 antagonistic mechanism that regulates pacemaker/working myocardial cell fate decision in the developing venous pole in mice, particularly in the junction domain of the sinoatrial node (SAN) and pulmonary vein myocardium where Shox2 and Nkx2-5 are co-expressed. In such regulatory mechanism, the stronger transcription output by Shox2 promotes pacemaker cell fate, while stronger Nkx2-5 output favors working myocardial fate. Interestingly, we also found that a majority of Hcn4-positive cells derived from mouse embryonic stem (mES) cells co-express Shox2 and Nkx2-5, suggesting that in vitro differentiated pacemaker-like cells also adopt such an antagonistic mechanism to regulate cell fate. To establish an in vitro modeling system to further dissecting the functional importance of the Shox2-Nkx2-5 antagonism and for future in vitro differentiation of pacemaker cells, we established induced pluripotent stem (iPS) cell lines from mice carrying allelic series of Shox2 and Nkx2-5, as well as lineage specific Cre and RosamTmG reporter alleles. These iPS cell lines are being tested to prove the principle that the Shox2-Nkx2-5 antagonistic mechanism also functions in iPS cell-derived cardiomycytes to regulate cell fate, and are being used for lineage tracing of differentiated cardiomycytes. Furthermore, these cell lines could also be applied to modeling human diseases with Nkx2-5 mutations in vitro.