Multiple gene transfections are often required to control the differentiation of embryonic stem cells. This is typically done by removing the cells from the culture substrate and conducting gene transfection via bulk electroporation (in suspension), which is then followed by further culture. Such repetitive processes could affect the growth and behavior of delicate/scarce adherent cells. We have developed a novel nanofiber-based sandwich electroporation device capable of in situ and in culture gene transfection. Electrospinning was used to deposit poly(epsilon-caprolactone)/gelatin nanofibers on the Al2O3 nanoporous support membrane, on top of which a polystyrene microspacer was thermally bonded to control embryonic stem cell colony formation. The applicability of this system was demonstrated by culturing and transfecting mouse embryonic stem cells. Measurements of secreted alkaline phosphatase protein and metabolic activity showed higher transfection efficacy and cell viability compared to the conventional bulk electroporation approach.