Bone marrow (BM) mesenchymal stem cells (MSCs) are non-hematopoietic cells capable of generating colonies of plastic-adherent marrow mesenchymal cells, each derived from a single cell termed a colony-forming unit fibroblasts (CFU-Fs). In addition to their role in establishing the marrow microenvironment, these cells have been shown to differentiate into several types of mesenchymal and non-mesenchymal lineages. Because of their multipotency, MSCs represent an attractive cellular source in the promising field of cellular therapy. In this chapter, we will focus on culture conditions for human BM MSC expansion and CFU-F assays. We also describe the methodologies to analyze the primary cultures obtained, both at the phenotypic and at the functional levels. Phenotypically, MSCs can be defined with a minimal set of markers as CD31-, CD34-, and CD45-negative cells and CD13-, CD29-, CD73-, CD90-, CD105-, and CD166-positive cells. Functionally, we describe the culture conditions (specific media and cellular preparations) for in vitro differentiation of MSCs into the adipogenic, osteogenic, and chondrogenic lineages. The corresponding colorimetric assays (oil red O, Von Kossa and alizarin red S, and safranin O and alcian blue stains, respectively) are also described.