Cells of the central nervous system are notoriously difficult to transfect. This is not only true for neurons and glial cells but also for dividing neural stem and progenitor cells (NSCs). About ten years ago a major advance was provided by introduction of the nucleofection technology that allowed for transfection of approximately half of the exposed NSCs. However, limitations were encountered with the need for large numbers of NSCs for a single transfection and compromised survival rates with typically only one-third of the cells surviving the pulse conditions. Here, we report the establishment of a pulse protocol that targets NSCs with high efficiency and twofold higher NSC survival rates using the 4D Nucleofector device. We demonstrate that the established protocol not only provides a clear and significant improvement over existing protocols with transfection rates above 80% and two-thirds of the NSCs surviving for at least 48h, but also their unaltered differentiation along neuronal and glial lineages. This improved protocol for the transfection of sensitive mouse central nervous system derived cells will provide an important step forward for studies of gene function by overexpression or knock-down of genes in cultured NSCs.