Mesenchymal stem cells, now commonly called mesenchymal stromal cells (MSCs), represent a rare and heterogeneous population of stem/progenitor cells that reside primarily in the bone marrow (BM) but can also be isolated from different sites such as adipose tissue, peripheral blood, cord blood, liver and fetal tissues. They are described as non-hematopoietic, clonogenic, plastic-adherent cells and capable of differentiating into multiple mesodermal lineages. They lack the inherent ethical consideration of embryonic stem cells and thanks to their capacity to home and engraft into injured tissues and modulate immune response, cell survival and angiogenesis, mesenchymal stromal cells represent a potential novel candidate for regenerative medicine. MSC immune-modulating properties make also them tools for the treatment of graft-versus-host disease (GVHD) as well as severe auto-immune diseases. This clinical interest needs to be substantiated by a clear understanding of the MSC molecular and cellular mechanisms, leading to their use in human therapeutics in the best conditions of security and efficacy. This chapter first presents what is actually known about MSC phenotypic and functional characteristics, focusing on their differentiation, trophic and immune-modulatory capacities. It then briefly describes the prerequisites for MSC culture in vitro for clinical applications.