Objective: We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly based on the frequent medium change in primary culture and diminishing the trypsinization time. Materials and Methods: Mouse mesenchymal stem cells (mMSCs) are generally isolated from an aspirate of bone marrow harvested from the tibia and femoral marrow compartments, then cultured in a medium with DMEM and FBS, for 3 hours in a 37°C-5% CO2 incubator. Non-adherent cells are carefully removed after 3 hours and fresh medium is replaced. When primary cultures become nearly confluent, the culture is treated with 0.5ml of 0.25% Trypsin containing 0.02% EDTA for 2 minutes at room temperature. For confirmation mesenchymal nature, the cells were induced to differentiate along osteoblastic, adipogenic, chondrogenic. Furthermore, the expression of some surface antigens was investigated. Results: Cells isolated using this method demonstrated the MSCs characteristics including their ability to differentiate into mesenchymal lineages. The cells retained the differentiation potentials in expanded cultures up to 10 passages. The cells were reactive to the CD44, Sca-1, and CD90 cell surface markers. The cells were negative for the hematopoietic surface markers such as CD34, CD11b, CD45, CD31, CD106, CD117 and CD135. Conclusion: A purified population of MSCs can be obtained three weeks after the initiation of culture.