Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis approaches used in the study of embryonic development and disease conditions. This chapter deals with the maintenance and modification of these pluripotent cell lines and describes the routes that can be taken for their efficient introduction to the in vivo environment.