Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the β-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated virus (AAV) inverted terminal repeats (Ad.LCR-β-GFP). This vector possesses the adenovirus type 35 fiber knob that allows efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth factor-dependent CD34⁺ erythroleukemic cell line, and in cord blood-derived human CD34⁺ cells. Stable transduction of MO7e cells with Ad.LCR-β-GFP was more efficient and less subject to position effects and silencing than transduction with a vector that did not contain the β-globin LCR. Analysis of integration sites indicated that Ad.LCR-β-GFP integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed integration sites were within the chromosomal β-globin LCR. None of the Ad.LCR-β-GFP integrations occurred in exons. The integration pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the β-globin LCR-containing vector. Infection of primary CD34⁺ cells with Ad.LCR-β-GFP did not affect the clonogenic capacity of CD34⁺ cells. Transduction of CD34⁺ cells with Ad.LCR-β-GFP resulted in vector integration and erythroid lineage-specific GFP expression.