The silencing of retrovirus vectors in stem cells poses a problem for their use in gene therapy and for studying stem cell biology. The identification of retrovirus silencer elements has led to the development of vectors with improved expression but these vectors are still susceptible to silencing in stem cells. DNase I footprinting across the entire MSCV retrovirus vector was performed, comparing F9 and NIH3T3 cell nuclear extracts. The results revealed differences in footprints and hypersensitive bases within the essential U5 and psi regions. Electrophoretic mobility shift assays were carried out to confirm binding and to identify the consensus sites through mutational analysis. Functional characterization of the U5 and psi regions using an episome repression assay identified a novel stem cell specific potential silencer element within the psi region.