Recent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus–based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1α and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1α promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1α promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3′ long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.