Transcriptional silencing of retrovirus vectors in stem cells is a major obstacle to current gene therapy approaches. Insulator elements can overcome this problem by protecting transgenes from position effects. The best studied insulator, the chicken β globin locus (5 HS4), has to date had limited effectiveness in viral vectors. A novel insulator has been discovered within the D4Z4 repeat sequence that is involved in human Facio-scapulo-humeral dystrophy (FSHD). We evaluate here the effect of D4Z4 on the silencing of retrovirus vectors in mouse embryonic stem (ES) cells. We used the HSC1 self-inactivating retrovirus vector containing the eGFP reporter gene controlled by the human EF1α promoter. This vector was partially insulated by two core HS4 insulators in the LTRs, and the D4Z4 insulator was added between gag and the EF1α promoter. Mouse J1 ES cells and NIH3T3 fibroblasts were infected at low MOI to obtain one provirus copy per cell. However, silencing and the potential effect of D4Z4 were obscured by the large non-infected cell population. To overcome this problem, we incorporated an eGFP-puromycin bicistronic gene into our vectors. Infected cells were quickly selected with puromycin and then maintained without further selection for long term eGFP expression detection by flow cytometry. We observed silencing of HSC1 with or without HS4 insulators in the LTR. In contrast, the D4Z4 vector was protected from silencing over time. These results indicate that the D4Z4 insulator enhances stable expression of the HSC1 vector in stem cells and is well suited for stem cell gene therapy applications.