RNA interference is widely used for loss-of-function studies in mammalian cells. As an alternative to the transfection of small RNAs, plasmid vectors have been developed to express short hairpin RNAs (shRNAs). We engineered the pHYPER shRNA vector, which is based on a 2.5-kb mouse genomic fragment encompassing the H1 gene. We have previously shown that this shRNA vector is highly efficient for both transient transfection studies in embryonic stem (ES) cells and generation of stable ES cell lines. Following ES cell transfection, the H1 promoter of pHYPER is recognized by the RNA polymerase III machinery, which directs the transcription of the shRNA. We provide here detailed protocols that explain how to optimize the use of pHYPER in ES cells.