The isolation and characterization of embryonic and adult stem cells from higher-order mammalian species will enhance the understanding of the biology and therapeutic application of stem cells. The aim of this study was to purify rhesus mesenchymal stem cells (MSCs) from adult bone marrow and to characterize functionally their abilities to differentiate along diverse lineages. Adherent cells from adult rhesus macaque bone marrow were characterized for their growth characteristics, lineage differentiation, cell-surface antigen expression, telomere length, chromosome content, and transcription factor gene expression. Rhesus bone marrow MSCs (BMSCs) are very heterogeneous, composed of primarily long, thin cells and some smaller, round cells. The cells are capable of differentiating along osteogenic, chondrogenic, and adipogenic lineages in vitro. The cell morphology and multipotential differentiation capabilities are maintained throughout extended culture. They express CD59, CD90 (Thy-1), CD105, and HLA-1 and were negative for hematopoietic markers such as CD3, CD4, CD8, CD11b, CD13, CD34, and platelet endothelial cell adhesion molecule-1 (CD31). BMSCs were also demonstrated to express the mRNA for important stem cell-related transcription factors such as Oct-4, Sox-2, Rex-1, and Nanog. Rhesus BMSCs have a normal chromosome content, and the shortening of telomeres is minimal during early passages. These data demonstrate that BMSCs isolated from rhesus macaques have a high degree of commonality with MSCs isolated from other species. Therefore, isolation of these cells provides an effective and convenient method for rapid expansion of pluripotent rhesus MSCs.