Research applications and cell therapies involving genetically modified cells require reliable, standardized and cost-effective methods for cell manipulation. The goal of this work is to provide a novel methodology that produces, in a single standardized techonology, genetic modification and cell isolation. We have named this novel procedure ―Magselectofection”. The approach is based on magnetic cell separation and magnetically-guided gene delivery (magnetofection). Optimized gene vectors associated with novel magnetic nanoparticles were formulated to transfect/transduce target cells while they are passaged and separated through a high gradient magnetic field cell separation column. Magnetofection of the Jurkat T cells using selected vector formulations resulted in a significant (up to 4.5-fold) enhancement in both luciferase reporter gene expression and the percentage of cells expressing eGFP, as compared to lipofection. A procedure for vector loading on LS Miltenyi columns was developed that enables up to 100% retention for both non-viral and viral magnetic complexes. We demonstrate, using a model cell mixture of K562 and Jurkat T cells, that the integrated method is highly efficient and specific for the target cell population. This was not only true for the model Jurkat/K562 mixture, but also for Sca-1+ mouse hematopoietic stem cells. With human umbilical cord mesenchymal stem cells (hUC-MSCs), we achieve up to 30% transfected cells with non-viral vector doses as low as 8 pg plasmid DNA per cell and up to 100% transduced cells with a multiplicity of infection of 0.5 TU/cell using lentivirus. Similarly, we obtain 22% eGFP-positive human cord blood hematopoietic stem cells (hCB-HSCs) upon lentiviral magselectofection compared to 0.15% eGFP-positive cells post-standard infection. We achieve up to 50% transduced Sca-1+ mouse stem cells at a lentiviral MOI of 1-3. Up to 5-15% and 20% genetic modified PBMC were found using non-viral and viral magselectofection, respectively. After genetic modification using magselectofection differentiation potential of hCB-HSCs and hUC-MSCs was maintained. Magselectofection requires a minimal number of manipulation steps and results in efficient and specific gene delivery to target cells. This minimizes the necessary vector material while maintaining the cellular differentiation potential of modified stem cells. Magselectofection may become a useful tool for nucleic acid therapy approaches involving ex-vivo genetically modified cells.