The bone marrow harbors a population of mesenchymal stem cells (MSCs) that possess the potential to differentiate into bone, cartilage, and fat, and along other tissue pathways. To date, MSCs from various species have been studied. Despite the bovine experimental model being widely used in experiments in vivo and in vitro, only a limited amount of information regarding bovine MSCs is available. The aim of this study was to isolate and induce the multilineage mesenchymal differentiation of bovine MSCs, thereby initiating further research on these cells. Bovine MSCs were isolated from eight calves, and osteogenic, chondrogenic, and adipogenic differentiation was induced by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by a quantitative “real time” reverse transcription/polymerase chain reaction technique. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells and formed alkaline-phosphatase-positive colonies; during differentiation, these colonies transformed into mineralized nodules. In addition, osteogenesis was followed by osteocalcin and collagen type I mRNA expression. Chondrogenesis was confirmed by the demonstration of collagen type II, aggrecan, and sox9 mRNA expression in the cells stimulated by transforming growth factor β1 in monolayer culture. After being cultured in an adipogenesis-inducing medium, the MSCs responded by the accumulation of lipid vacuoles and the expression of adipocyte-specific genes. We have therefore demonstrated that cells harvested from bovine bone marrow are capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research.