Embryonic stem (ES) cells are pluripotent cells derived from the epiblast of preimplantation embryos. These cells are emerging as a key model system for elucidating mechanisms involved in development and disease as well as having a unique potential as a source of unlimited somatic cells for transplantation therapies. ES cells can be easily manipulated at the DNA level, allowing both transient and stable expression of complementary DNA encoding transgenes of interest. The human cytomegalovirus (CMV) immediate-early enhancer and promoter is commonly used for transient expression of transgenes in ES cells. However, its use in the formation of stable cell lines is less common. We demonstrate an electroporator transformation technique that results in up to 90% transfection efficiency of CMV-encoding vectors in ES cells. Furthermore, we describe the design of vectors and cloning techniques that allow stable expression of transgenes under control of the CMV promoter and a fluorescent microscopy method for detecting protein expression in ES cells in situ.