Vectors derived from human immunodeficiency virus (HIV) hold promise for efficient gene delivery into human hematopoietic cells. In this study, HIV vectors containing different combinations of cis-acting elements, including the HIV central flap sequence, and the woodchuck posttranscriptional regulatory element (WPRE) in combination with two different promoters, were used to transduce primary human lymphocytes and cord blood CD34+ progenitor cells. The effect of these elements on the transduction efficiency and transgene expression was systematically evaluated. The results demonstrate that with the combination of flap, WPRE sequences, and the promoter derived from spleen focus-forming virus (SFFV), a foreign gene can be efficiently delivered into primary human T lymphocytes and cord blood CD34+ cells. The study establishes the parameters for proper vector design to efficiently deliver foreign genes into human hematopoietic cells.