BACKGROUND: Nucleus free red blood cells are unique to mammals. During their terminal stage of differentiation, mammalian erythroblasts exit the cell cycle and enucleate. We previously found that survivin, a member of the chromosomal passenger complex that is required for cytokinesis, is highly expressed in late non-dividing cells. The role of survivin in enucleating erythroblasts is not known. DESIGN AND METHODS: In order to identify the role of survivin in these late erythroblasts, we performed proteomic analysis on survivin-bound protein complexes purified from murine erythroleukemia cells. Various molecular and cell biological techniques were used to confirm the presence and function of this novel complex. Furthermore, we used survivin(fl/fl) mice to study the effect of loss of survivin in enucleating erythroblasts. RESULTS: We found that survivin failed to co-localize with its known partners' inner centromere protein or Aurora-B in enucleating erythroblasts but rather exists in a multi-protein complex with epidermal growth factor receptor substrate15 and clathrin, two proteins that mediate endocytic vesicle trafficking. As evidence for a direct role of this latter complex in enucleation, we found that knockdown of the genes reduced the efficiency of enucleation of primary human erythroblasts. We also observed that loss of survivin in murine erythroblasts inhibited enucleation and that survivin-deficient cells harbored smaller cytoplasmic vacuoles. Interestingly, vacuolin-1, a small molecule that induces vacuole fusion, rescued the defective enucleation caused by survivin deficiency. CONCLUSIONS: This study identified a novel role for survivin in erythroblast enucleation through previously unknown protein partners.