Human immunodeficiency virus type 1 (HIV-1) replication in T cells can be inhibited by RNA interference (RNAi) through short hairpin RNA (shRNA) expression from a lentiviral vector. However, for the development of a durable RNAi-based gene therapy against HIV-1, multiple shRNAs need to be expressed simultaneously in order to avoid viral escape. In this study, we tested a multiple shRNA expression strategy for different shRNAs using repeated promoters in a lentiviral vector. Although highly effective in co-transfection experiments, a markedly reduced activity of each expressed shRNA was observed in transduced cells. We found that this reduced activity was due to recombination of the expression cassette repeat sequences during the transduction of the lentiviral vector, which resulted in deletions of one or multiple cassettes. To avoid recombination, we tested different promoters for multiple shRNA expression. We compared the activity of the human polymerase III promoters U6, H1, and 7SK and the polymerase II U1 promoter. Activities of these promoters were similar, irrespective of which shRNA was expressed. We showed that these four expression cassettes can be combined in a single lentiviral vector without causing recombination. Moreover, whereas HIV-1 could escape from a single shRNA, we now show that HIV-1 escape can be prevented when four shRNAs are simultaneously expressed in a cell.