Optimization of Nucleofection: six programs (A-006, A-012, A-013, A-023, A-027, B-016), two solutions, and two cell harvesting methods (collagenase, trypsin) were tested. Best results were obtained by using trypsin-derived single cells with program A-023 (transfection efficiency 61 %), and these conditions were used in all following experiments. 2x10^6 hES cells (H1 or H9 cell lines) were trypsinized, resuspended in 100 µL mES solution plus 2-3 µg DNA (or shRNA, siRNA). Nucleofection of pmaxGFP or 4.9kb vector containg hrGFP (GFP-neo) resulted in efficiencies with a mean of 76 %. Stable GFP-neo expression (selection by G418) achieved 85 % efficiency. RNAi: Transfection of shRNA expressing vector or siRNA (co-transfected with pmaxGFP) against Oct 4 or Nanog; analysis by RT-PCR with beta-2M gene as control. Data strongly support that nucleofection of siRNA/shRNS vectors effectively alters gene expression. Stable introduction of AP-specific shRNA reduces expression of the gene without effecting hES cell characteristics. Altogether, using nucleofection combined with culture conditions improving ES cell survival, transient transfection efficiencies of up to 85 % and a stable transfection efficiency of up to 1.2 in 10^4 cells was achieved.