During the past 7 years, RNA interference (RNAi) has emerged as a powerful gene-knockdown technology in many model eukaryotic systems because of the functional conservation of the RNAi machinery, and the ease with which exogenous double-stranded RNA (dsRNA) triggers of gene silencing can be delivered into cultured cells and even whole organisms. In mammalian cells, large-scale RNAi screens have now been carried out using both transient RNAi triggers, such as small interfering RNAs (siRNAs) and stably expressed short hairpin RNAs (shRNAs). One area of particular interest in mammals is the application of RNAi in embryonic stem (ES) cells, where RNAi can be used for functional genetic studies and also for specifying particular cell types needed for cell-replacement therapies. Here we report a simple yet effective method for transfecting into mouse ES cells RNA interference triggers, in the form of in vitro synthesized siRNAs or synthetic shRNA3 (Fig. 1). Using this protocol to introduce synthetic shRNAs3 into a Nanog-GFP reporter cell line, we routinely achieve >85% knockdown for target genes. This procedure is readily 'scalable' for high-throughput applications and can be adapted for the delivery of siRNAs, enzymatically cleaved dsRNAs6 and plasmids encoding expressed shRNAs. The protocol describes transfection of mouse ES cells in 24-well tissue culture plates. It can be modified to accommodate any well size by simply scaling the amount of RNA, the number of cells and the volume of medium according to the relative surface area of the well.