Gene trapping can be used to introduce insertional mutations into the genome of mouse embryonic stem cells (ESCs). The method has been adapted for high-throughput use, in an effort to inactivate all genes in the mouse genome. Gene trapping is performed with vectors that simultaneously inactivate and report the expression of the trapped gene and provide a molecular tag for its rapid identification. Gene-trap approaches have been used successfully in the past by both academic and commercial organizations to create libraries of ESC lines harboring mutations in single genes that can be used for making mice. Presently, approximately 70% of the protein-coding genes in the mouse genome have been disrupted by gene-trap insertions. Here we describe the basic methodology used to induce and characterize gene-trap mutations in ESCs.