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We compared the viability, proliferation, and differentiation of human Mesenchymal Stromal Cells (MSC) after culture on poly(lactic-co-glycolic acid) (PLGA) and PLGA/chitosan (PLGA/CH) hybrid scaffolds. We applied conventional and emulsion electrospinning techniques, respectively, for the fabrication of the PLGA and PLGA/ CH scaffolds. Electrospinning under optimum conditions resulted in an average fiber diameter of 166 ± 33 nm for the PLGA/CH and 680 ± 175 nm for the PLGA scaffold. The difference between the tensile strength of the PLGA and PLGA/CH nanofibers was not significant, but PLGA/CH showed a significantly lower tensile modulus and elongation at break. However, it should be noted that the extensibility of the PLGA/CH was higher than that of the nanofibrous scaffolds of pure chitosan. As expected, a higher degree of hydrophilicity was seen with PLGA/ CH, as compared to PLGA alone. The biocompatibility of the PLGA and PLGA/CH scaffolds was compared using MTS assay as well as analysis by scanning electron microscopy and confocal microscopy. The results showed that both scaffold types supported the viability and proliferation of human MSC, with significantly higher rates on PLGA/ CH nanofibers. Nonetheless, an analysis of gene expression of MSC grown on either PLGA or PLGA/CH showed a similar differentiation pattern towards bone, nerve and adipose tissues.