Large animal models have played a crucial role in the development of gene therapy protocols. A significant advantage of large animal models over rodent models includes the ability to more easily translate protocols developed in large animals to humans. For gene therapy applications, nonhuman primates and canines have been the main large animal models. Canines have the advantage that there are disease models available, e.g., hemolytic anemia (pyruvate kinase deficiency), leukocyte adhesion deficiency, severe combined immunodeficiency (XSCID), storage diseases, and others. In addition, all three major integrating virus systems, i.e., gammaretrovirus-, HIV-derived lenti- and foamy virus vectors are able to efficiently transduce canine hematopoietic cells. Here we describe protocols developed for efficient transduction of canine hematopoietic repopulating cells.