Despite several strategies developed for replenishing the dead myocardium after myocardial infarction (MI), stem cell therapy remains the leading method to regenerate new myocardium. Although induced pluripotent stem cells (iPS) and transdifferentiation of the differentiated cells have been used as novel approaches for myocardial regeneration, these approaches did not yield very successful results for myocardial regeneration in in vivo studies. Asynchronous contractility of newly formed cardiomyocytes with the existing cardiomyocytes is the most important issue with iPS approach, while very low yield of transdifferentiated cardiomyocytes and theirÂ less chances to beat in the same rhythm as existing cardiomyocytes in the MI heart are important caveats with transdifferentiation approach. CSCs are present in the heart and they have the potential to differentiate into myocardial cells. However, the number of resident CSCs is very low. Therefore, it is important to get maximum yield of CSCs during isolation process from the heart. Increasing the number of CSCs and initiating their differentiation ex vivo are crucial for CSC-based stem cell therapy. Here, we present a better method for isolation, characterization and differentiation of CSCs from the mouse heart. We also demonstrated morphological changes in the CSCs after 2Â days, 3Â days, and 7Â days in maintenance medium and a separate group of CSCs cultured for 12Â days in differentiation medium using Phase-Contrast microscopy. We have used different markers for identification of CSCs isolated from the mouse heart such as marker for mouse CSC, Sca-1, cardiac-specific markers NKX2–5, MEF2C, GATA4, and stemness markers OCT4 and SOX2. To characterize the differentiated CSCs, we used CSCs maintained in differentiation medium for 12Â days. To evaluate differentiation of CSCs, we determined the expression of cardiomyocyte-specific markers actinin and troponin I. Overall; we described an elegant method for isolation, identification, differentiation and characterization of CSCs from the mouse heart.