Gene targeting in mice has been used extensively to elucidate gene function in vivo. However, for gene targeting to be successful, the targeting vector must be carefully designed. This chapter addresses the rationale behind designing targeting vectors, detailing the essential components, and highlighting specific considerations for different types of vectors, from gene deletions to point mutations and insertions. Examples of vector designs, cloning strategies, and approaches for successful screening of recombinants are described. The use of Cre/LoxP and Flp/frt systems for conditional targeting is described, together with strategies for generating conditional deletions. Methods for generating conditional point mutations are also described and their potential drawbacks discussed.