Cell therapy with a variety of stem populations is increasingly being investigated as a promising regenerative strategy for cardiovascular (CV) diseases. Their combination with adequate scaffolds represents an improved therapeutic approach. Recently, several biomaterials were investigated as scaffolds for CV tissue repair, with decellularized extracellular matrices (dECMs) arousing increasing interest for cardiac tissue engineering applications. The aim of this study was to analyze whether dECMs support the cardiac differentiation of CardiopoieticAF stem cells. These perinatal stem cells, which can be easily isolated without ethical or safety limitations, display a high cardiac differentiative potential. Differentiation was previously achieved by culturing them on Matrigel, but this 3D scaffold is not transplantable. The identification of a new transplantable scaffold able to support CardiopoieticAF stem cell cardiac differentiation is pivotal prior to encouraging translation of in vitro studies in animal model preclinical investigations. Our data demonstrated that decellularized extracellular matrices already used in cardiac surgery (the porcine CorTMPATCH and the equine MatrixPatchTM) can efficiently support the proliferation and cardiac differentiation of CardiopoieticAF stem cells and represent a useful cellular scaffold to be transplanted with stem cells in animal hosts.