Mesenchymal stem cells are typically enriched from bone marrow via their preferential attachment to tissue culture plastic. However, this isolation method has proven ineffective for murine MSCs because various hematopoietic cell lineages survive and/or proliferate on stromal layers in the absence of exogenous cytokines and therefore constitute a large percentage of the plastic adherent population. Although various methods have been described to remove contaminating hematopoietic populations from these cultures none have gained widespread acceptance. Consequently, we developed a method based on immunodepletion to fractionate hematopoietic and endothelial lineages from plastic adherent fibroblastoid (stromal) cells elaborated from murine bone marrow. Colony-forming assays, immunostaining and flow cytometry has been used to validate the effectiveness of this method. Moreover, immunodepleted populations have been shown to exhibit the capacity for multilineage differentiation in vitro and in vivo and therefore retain the characteristics of MSCs. Most recently, we also catalogued the transcriptome of immunodepleted populations via serial analysis of gene expression. Therefore, our immunodepletion scheme provides a means to enrich from murine bone marrow MSCs that's molecular and biological characteristics are well described. Importantly, this immunodepletion method does not employ long-term expansion of plastic adherent cells ex vivo, thereby avoiding the generation of immortalized cell lines.