The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. In order to achieve this, we used a 500 bp fragment from the proximal WASP gene promoter to drive the expression of the WASP cDNA in the context of a self-inactivating lentiviral vector. Single-round transduction of WASp-deficient herpesvirus saimiri (HVS)-immortalized cells as well as primary allospecific T cells from Wiskott-Aldrich syndrome (WAS) patients with this vector (WW) resulted in expression levels similar to those of control cells. Non-HCs were transduced with similar efficiency, but the levels of WASp were 135-350 times lower than those achieved in HCs. Additionally, transduction of WASp-deficient cells with WW conferred a selective growth advantage in vitro. Therefore, lentiviral vectors incorporating proximal promoter sequences from the WASP gene confer hematopoietic-specific, and physiological protein expression.