Human embryonic stem cells (HESCs) are widely used as a model system for human cell type specification. Genetic modification forms a valuable tool for HESC technology, as it provides the basis for lineage selection, i.e., the purification of a specific cell type after differentiation. Electroporation is an efficient way to transfect HESCs. Nucleofection is an electroporation-based transfection technique which utilizes cell-type-specific buffer solutions and specific electric settings. Customization of these two parameters has been proven to result in highly efficient gene transfer even in hard-to-transfect cells. We can show that nucleofection surpasses conventional electroporation in efficiency and decreases the experimental effort for transfection of HESCs.