p53 is one of the most well-characterized members of the tumor suppressor gene family. The role of p53 in controlling cellular homeostasis has proven critical, with over half of all human tumors having either lost or mutated p53 . The emergence of technology facilitating the ablation of a gene within an animal’s genome allowed great advances in the study of p53 . The p53 knockout mouse was one of the first of its kind and provided a powerful tool for the study of p53. Production of the p53 knock-out mouse demonstrated the protein’s dispensability during embryogenesis, while highlighting its essential role in controlling tumor formation. A variety of p53 mutant models have emerged since the original p53 knock-out mouse, along with improved techniques for regulating gene targeting. This chapter describes the necessary steps and protocols involved in producing a mutant mouse as well as the characterization that follows.