Because of its many favorable characteristics, the zebrafish has become a popular model for studies of vertebrate development (1 ,2 ). To further enhance the utility of the zebrafish model for the genetic analysis of embryogenesis, we have been working to establish methods for cell-mediated gene transfer and targeted mutagenesis using pluripotent zebrafish embryonic stem (ES) cells (3 –5 ). Successful ES cell-mediated gene transfer requires that the cultured pluripotent ES cells are able to produce viable germ cells after being introduced into a host embryo (6 ,7 ). ES cells are genetically altered in culture by the incorporation of foreign DNA, and, following in vitro selection and expansion, the cells are introduced into the host embryo where they participate in normal development and contribute to the germ cell lineage. When sexually mature, the germline chimeric animal possesses eggs or sperm derived from ES cells, making it possible to pass the genetic alteration on to subsequent generations to establish the transgenic or knockout line (6 ,7 ). This chapter describes a protocol for the derivation of germline-competent pluripotent zebrafish ES cell cultures, along with methods for introducing foreign DNA into cells by electroporation and for using the cultured ES cells to generate germline chimeric embryos.