The combined use of site-specific recombination and gene targeting or trapping in embryonic stem cells (ESCs) has resulted in the emergence of technologies that enable the induction of mouse mutations in a prespecified temporal and spatially restricted manner. Their large-scale implementation by several international mouse mutagenesis programs will lead to the assembly of a library of ES cell lines harboring conditional mutations in every single gene of the mouse genome. In anticipation of this unprecedented resource, this chapter will focus on site-specific recombination strategies and issues pertinent to ESCs and mice. The upcoming ESC resource and the increasing sophistication of site-specific recombination technologies will greatly assist the functional annotation of the human genome and the animal modeling of human disease.