Clinical trials utilizing bone marrow-derived mesenchymal stem cell (BM-MSC) therapies show promise for treating a variety of pathologic conditions. Paramount to optimization of such cell-based therapies is a thorough understanding of MSC biology. Despite the tremendous potential that exists for the clinical use of canine BM-MSCs in veterinary medicine, as well as in preclinical studies for human medicine, relatively little information exists regarding basic biological properties of the cells. In this study, we compared the importance of donor characteristics (age and harvest site) and ex vivo expansion on canine BM-MSC frequency (CFU-f) and differentiation potential. Advancing age was found to have a negative effect on CFU-f as well as osteogenic potential. Site of harvest was also found to have significant effects on MSC properties. MSCs obtained from the humerus were found at the lowest frequency and were least osteogenic compared to those harvested from the tibia, femur, and ilium. Osteogenic potential diminished significantly by the third passage. These results suggest important donor parameters and culture effects to consider in translational studies examining MSC-based regenerative medical strategies.