Objective: Human bone marrow mesenchymal stem cells (hMSCs) can differentiate into several types of mesenchymal cells, inducing osteocytes, adipocytes, and chondrocytes. Until now, many protocol for inducing chondrocyte-differentiation in hMSCs in vitro have been reported. In this study, we induced differentiation into chondrocyte phenotype in the MSCs population by new protocol. Our objective was to study the effects of Synovial Fluid on chondrogenic differentiation of hMSCs in culture. Synovial Fluid could promote expression of chondrogenic markers, Sox9 and collagen II mRNA. Chondrogenesis was induced by Synovial Fluid , which encourage tissue engineering applications of MSC in chondral defects, as the natural environment in the joint is favorable for chodrogenic differentiation. Materials and Methods: hMSCs (passage2) were plated in 6-well plates in DMEM containing 1% FBS .To induce chondrogenic differentiation, the cells were treated with different concentration of Synovial fluid in monolayer and micromass cultures. The expression of the Sox9 and Col2a1 was confirmed by RT-PCR and cells staining during 21days Results: Human MSCs cultured in monolayer and treated with SF exhibited a more rounded shape than the cells from the control group. The expression of chondrocyte-specific genes that occurred following SF treatment was found to be dose-dependent and time-dependent. We checked the expression of well-known cartilage-specific genes. An expression analysis for the known chondrogenic markers Sox9 and Col2a1 using RT-PCR in differentiated hMSCs was performed. In this manner, we showed that Sox9 and Col2a1 were induced during the course of hMSCs differentiaton into chondrogenic lineage . Conclusion: Our results showed that hMSCs treatment with 200µl/ml Synovial Fluid differentiated to chondrocytes . Phenotype of cells and gene expression changed during this differentiation. These data confirmed that MSCs can exhibit chondrocyte differentiation potential in vitro, depending on the protocols of inducement.