Tumor development is a complex and dynamic process that involves malignant, vascular, and stromal cells. Endosialin is a tumor endothelial marker (TEM) present in the microvasculature and stroma of human tumors. Cancer-associated fibroblasts (CAF) have been implicated in promoting tumor development and have been associated with mesenchymal stem cells (MSC). Since stem/progenitor cells recruited either from bone marrow or residing in nearby tissues can contribute to pathological processes we investigated endosialin in MSC using a novel monoclonal antibody. Endosialin is highly expressed by CAF and human bone marrow-derived MSC. MSC can form networks in a tube formation assay that is inhibited by an anti-endosialin antibody. Immunohistochemistry for human endosialin in xenograft tumors following co-injection of MSC and cancer cells identified MSC in tumor stroma. MSC are a potential target for anticancer therapeutic intervention and endosialin expression offers a new tool for the identification of MSC. Endosialin expression by both CAF and MSC further implies the potential contribution of MSC to tumor stroma via differentiation into tumor stromal fibroblasts.