Bone marrow (BM) contains a rare population of mesenchymal stromal cells (MSCs), which have been characterized as nonhematopoietic skeletal progenitor cells with central importance for the hematopoietic microenvironment. Classically, MSCs are isolated by plastic adherence and subsequent culture. However, as cultured stromal cells differ from their in vivo progenitors, it is important to identify the phenotype of the primary MSCs to study these cells in more detail. In the past years, several surface markers have been reported to be suitable for effective enrichment of BM‐MSCs, and recent data indicate that the putative MSC stem/progenitor cell population in human adult BM is highly enriched in Lin⁻ CD45⁻ CD271⁺ CD140a (PDGFRα)^low/− cells. Moreover, surface marker combinations have been described for the isolation of MSCs from murine BM. On the basis of these findings, the role of primary MSCs can now be studied in normal and, importantly, diseased BM. Furthermore, genetically engineered mouse models have been developed as powerful tools to investigate well‐defined BM stromal cell populations in vivo. Our discussion aims to provide a concise overview of the current state of the art in BM‐MSC isolation in humans and briefly present murine MSC isolation approaches and genetic models.