Tissue engineering is a technique that can be used for repairing damaged cartilage.The aim of this study was chondro-differentiation of mesenchymal stem cells (MSCs) on polyvinyl alcohol/gelatin/chondroitin sulfate (PVA/GT/CS) blend nanofibers. Nanofibers were fabricated by electrospinning method with different concentrations of CS (10%, 15%, and 20%). MSCs were seeded on nanofibers for biocompatibility and cell viability test by MTT assay. Alcian blue staining assay was done to show that MSCs were differentiated to chondrocyte by staining the glycosaminoglycans (GAGs) that was produced by cells. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) assay were done to confirm type II collagen expression. Scaffolds with different concentrations of CS were tested for biocompatibility, and the nanofiber with 15% of GAG showed better results (P = 0.02) compared with other nanofibers; therefore, it was chosen as the main sample for chondrogenesis tests. On the basis of scanning electron microscopy (SEM), hMSCs had a proper attachment to the nanofiber with 15% CS. Alcian blue staining showed the different rates of blue coloration, which means cells produced different types of GAGs from the GAG we used in the nanofiber. RT-PCR and ICC showed that COL2A1 was expressed in mRNA and protein levels. This study indicates that the PVA/GT/CS nanofiber containing 15% CS can be a good candidate for cartilage tissue engineering. K E Y W O R D S cartilage, chondroitin sulfate, electrospun, mesenchymal stem cells, tissue engineering