Over the last 30 years of biomedical research, technologies for transgene expression and gene loss of function have been developed from animal model systems to human gene therapy, and, increasingly, embryonic stem cells are a key target for these genetic engineering strategies. In this chapter, we describe how retrovirus/lentivirus vectors can be used to transfer and stably express genes or small interfering RNAs in mouse and human embryonic stem cells and their progeny, thereby allowing genetic gain-of-function or loss-of-function analysis and cell lineage selection in a multitude of experimental settings.