Pluripotent embryonic stem (ES) cell lines were first isolated over 25 years ago and remain an essential tool in molecular and developmental biology to this day. In particular, the use of homologous recombination and subsequent generation of ES-derived mice has greatly facilitated research across all fields. Moreover, ES cells represent an extremely attractive model to study events in early development. In this chapter, we will describe the derivation and propagation of murine ES cells. This is followed by a description of targeting ES cells and a protocol for the generation of mice by diploid and tetraploid blastocyst injections.