This chapter focuses on mesenchymal stem cells (MSCs), a multipotent stem cell type that has been found in a variety of tissues and organs of the human body since their discovery in 1970. Their main function is to maintain and repair the respective tissue in vivo. Mesenchymal stem cells can be easily isolated from different tissues and can undergo extensive self-proliferation prior to differentiation into various mesodermal cell types such as osteoblasts, chondrocytes, adipocytes, tenocytes, myocytes, and fibroblasts. Because of this vast differentiation potential, mesenchymal stem cells are a promising tool for regenerative medicine approaches. They could play an important role in cellular therapy, tissue replacement and regeneration in the future. Mesenchymal stem cells will be compared for their application and differentiation potential to embryonic stem cells and induced pluripotent stem cells and the limitations and challenges using scaffolds for tissue repair will be presented. In addition, legal and ethical aspects of the use of mesenchymal stem cells will be discussed. Moreover, isolation protocols for mesenchymal stem cells from the most common sources namely bone marrow, adipose tissue and umbilical cord are included.