Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities. The establishment of methods for large-scale expansion of clinical-grade MSCs in vitro has paved the way for their therapeutic use in clinical trials. However, the clinical application of MSCs also requires cryopreservation and banking of the cell products. To preserve autologous or allogeneic MSCs for future clinical applications, a reliable and effective cryopreservation method is required. Developing a successful cryopreservation protocol for clinical stem cell products, cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate are all aspects which should be considered. A major challenge is the selection of a suitable cryoprotectant which is able to penetrate the cells and yet has low toxicity. This chapter focuses on recent technological developments relevant for the cryopreservation of MSCs using the most commonly used cryopreservation medium containing DMSO and animal serum or humanderived products for research use and the animal protein-free cryopreservation media CryoStor (BioLife Solutions) for clinical use.