Lentiviral vectors efficiently transduce quiescent stem cells and are being evaluated for gene therapy of blood dis-orders. The risk of genotoxicity as a result of insertional mutagenesis is an important safety consideration. The hy-persensitive site 4 insulator from the chicken β-globin locus (cHS4) possesses chromatin bar-rier and enhancer-blocking functions. A control lentiviral vector encoding green fluorescent protein was compared with a vector in which the cHS4 insulator element flanked the green fluorescent protein expression cassette in single cell isolates of transduced human T cells (Jurkat) after 9 days in culture. The insulator had minimal effect on mean fluorescent intensity and only modestly reduced the variability of green fluorescent protein expression among indi-vidual single cell isolates. Most unique integration sites were within genes, but the insulator-containing vector had a moderate predilection to integrate near the transcriptional start site compared with the control vector. Clonal domi-nance developed in cultures of cells containing the integrated vector genomes, as reflected by the recovery of mul-tiple single cell isolates containing the same integration site. We infer that certain integrations conferred a prolifera-tive or survival advantage by affecting gene expression through insertional mutagenesis, leading to this clonal dominance. This effect was diminished by including the insulator element in the vector genome.