Innovative approaches for the treatment of rare inherited diseases are hampered by limited availability of patient derived samples for preclinical research. This also applies for the evaluation of novel vector systems for the gene therapy of monogenic hematological diseases like X-linked chronic granulomatous disease (X-CGD), a severe primary immunodeficiency caused by mutations in the gp91(phox) subunit of the phagocytic NADPH oxidase. Since current gene therapy protocols involve ex vivo gene modification of autologous CD34(+) hematopoietic stem cells (HSC), the ideal preclinical model should simulate faithfully this procedure. However, the low availability of patient-derived CD34(+) cells limits the feasibility of this approach. Here, we describe a straightforward experimental strategy that circumvents this limitation. The knock down of gp91(phox) expression upon lentiviral delivery of shRNAs into CD34(+) cells from healthy donors generates sufficient amounts of X-CGD CD34(+) cells which subsequently can be used for the evaluation of novel gene therapeutic strategies using a codon-optimized gp91(phox) transgene. We have used this strategy to test the potential of a novel gene therapy vector for X-CGD.