Significance Despite intense interest over a period of decades, mechanisms of allelic exclusion have remained unsolved mysteries in the field of eukaryotic gene expression control. Parasitic African trypanosomes express variant surface glycoproteins (VSGs) in a monoallelic fashion and have long been a paradigm for studies in this area. We used an RNA interference screen for loss of exclusion and identified and characterized VSG exclusion 1 (VEX1). VEX1 sequestration restricts expression and prevents the simultaneous establishment of more than one active VSG gene. VEX1 also appears to reinforce sequestration-based exclusion through homology-dependent repression. Our results indicate a “winner-takes-all” mechanism that allows parasitic trypanosomes to express just one VSG gene at a time.