The generation of gene-targeted and transgenic mouse models facilitates the in vivo study of mammalian gene function. Advances in technologies to engineer the mouse genome have extended the choice of gene manipulation from straightforward gene inactivation or overexpression to detailed modification of gene expression pattern, structure, and function in desired cell types and at specific times. Combining conventional/conditional, knockout/knockin, inducible, and even reversible gene manipulation strategies provides the investigator with the freedom to design an optimal model to study the function of a gene in a specific organ system during development or in postnatal life. To maximize success, however, the requirements and limitations of each approach need to be considered. This chapter provides an overview of gene targeting strategies that are available for manipulation of the mouse genome. We emphasize approaches that aid the investigation of the development and function of the hematopoietic system in the mouse.