With the development of the Cre-LoxP system, conditional gene targeting has rapidly become a powerful technology that facilitates the study of gene function. This advanced technique circumvents three major concerns sometimes levelled against conventional transgenic and gene-targeting approaches. First of all, gene ablation may exert its effect in multiple cell and tissue types, creating a complex phenotype in which it is difficult to distinguish direct function in a particular tissue from secondary effects resulting from altered gene function in other tissues. Secondly, a gene deletion expressed in the germ line may cause embryonic lethality, thereby precluding analysis of gene function in the adult tissues. Thirdly, the transgenic approach represents a somewhat surreal over-expression of a given protein often causing spurious phenotypes. The generation of conditional knockout mice is a multiple-step process, which involves mating the flox mutant mouse line (essential exon/s of the gene of interest are flanked by two LoxP sites) and the Cre-expressing mouse line. Over the past few years many inducible and/or tissue-specific Cre mouse lines have been developed. This chapter will give a brief review of the generation of Cre-expressing mouse lines and will discuss the strategy of using these Cre lines. In addition, information regarding established Cre-expressing mouse lines will be provided.