Over the last decade, cell therapy has emerged as a potentially new approach for the treatment of cardiovascular diseases. Among the wide range of cell types and sources, adipose-derived mesenchymal stem cells have shown promise, mainly due to its plasticity and remarkable paracrine-secretion capacity, largely demonstrated at the in vitro and in vivo levels. Furthermore, its accessibility and abundance, the low morbidity of the surgical procedure, its easy isolation, culture, and long-term passaging capacity added to its immunomodulatory properties that could allow its allogeneic transplantation, making it one of the most attractive candidates for clinical application. In this chapter, we will focus on the methodology for the isolation, expansion, phenotypical characterization, differentiation, and storage of the adipose-derived stem cells.