Abstract The ability to effectively transduce human hematopoietic stem cells (HSCs) and to ensure adequate but “physiological” levels of transgene expression in different hematopoietic lineages represents some primary features of a gene-transfer vector. The ability to carry, integrate, and efficiently sustain transgene expression in HSCs strongly depends on the vector. We have constructed lentiviral vectors (LV) containing fragments of different lengths of the hematopoietic-specific regulatory element of the Wiskott-Aldrich syndrome (WAS) gene—spanning approximately 1,600 and 170 bp—that direct enhanced green fluorescent protein (EGFP) expression. The performance of vectors carrying the 1,600 and 170 bp fragments of the WAS gene promoter was compared with that of a vector carrying the UbiquitinC promoter in human cord blood CD34+ cells and their differentiated progeny both in vitro and in vivo in non-obese diabetic mice with severe combined immunodeficiency. All vectors displayed a similar transduction efficiency in CD34+ cells and promoted long-term EGFP expression in different hematopoietic lineages, with an efficiency comparable to, and in some instances (for example, the 170-bp promoter) superior to, that of the UbiquitinC promoter. Our results clearly demonstrate that LV containing fragments of the WAS gene promoter/enhancer region can promote long-term transgene expression in different hematopoietic lineages in vitro and in vivo and represent suitable and highly efficient vectors for gene transfer in gene-therapy applications for different hematological diseases and for research purposes. In particular, the 170-bp carrying vector, for its reduced size, could significantly improve the transduction/expression of large-size genes. Disclosure of potential conflicts of interest is found at the end of this article.