Epicardium is the outer cell layer of the heart. Its integrity and function are essential for normal heart development. To study the role of epicardium in both fetal and adult hearts, it is desirable to isolate and culture pure populations of these cells. Here we describe methods with Cre-loxP technology to lineage tag epicardial cells (EpiCs) and epicardium-derived cells (EPDCs), dissociate and isolate them by flow-activated cytometry sorting (FACS), and characterize them by quantitative PCR and immunostaining. This platform allows further characterization and manipulation of EpiCs and EPDCs for expression studies and functional assays.