Targeted gene manipulation has been used in the last few decades for better understanding of gene function. Most often mutant or overexpression genotypes are analyzed, but in many cases these are not sufficient to obtain a detailed picture on the mode of action of the corresponding protein. For example, many mutations result in pleiotropic or early phenotypic effects thereby affecting the whole organism. Conditional complementation or deletion of the gene under study in a specific cell or tissue can elucidate its exact role in a specific region within a certain time frame. Implementation of several site-specific recombination systems such as CRE/lox has created powerful tools to study the role of many genes at the cellular level. In this chapter, we describe in detail protocols for the application of a two-vector based CRE/lox system, enabling controlled timing and position of gain or loss of function clonal analyses.