The recent impetus in stem cell research has highlighted the potential use of bone marrow mesenchymal stem cells (MSC) for tissue regeneration. While little is known about their properties in situ, MSCs are thought to possess a "plasticity" that allows them to differentiate into a variety of different stromal cell types including bone, fat and cartilage, and other lineages such as muscle cells and astrocytes. However, studies have shown that only a minor proportion of MSCs clonal cell lines can maintain a primitive multi-potential phenotype following ex vivo expansion. Moreover, with successive subculture, progeny of MSCs display a diminished capacity to proliferate and differentiate into various tissues such as bone. This has hampered the use of MSCs in the development of cellular therapies, in particular for skeletal tissue regeneration. Our research team has developed an immunoselection protocol to purify human MSCs populations directly from bone marrow aspirates, based on their expression of the stromal precursor cell marker, STRO-1 and the vascular/smooth muscle antigens, CD106 and CD146. This methodology has allowed us to characterise the in vivo properties of purified human MSCs including the genotypic profile of this population. Several distinguishing features of freshly isolated MSCs suggest that important markers associated with “stemness” are rapidly lost in vitro, significantly impacting on their survival, life span and cell fate determination. Understanding the properties of stem cells in situ will help elucidate the fundamental conditions necessary to maintain and expand primitive MSC populations ex vivo, in order to effectively direct and enhance their developmental potential for a range of tissue engineering and gene therapy strategies.