To solve the shortage of blood sources has become an urgent need in the current medical. One of the important solutions is functional production of erythrocytes in vitro using stem/progenitor cells. But enucleation remains one of the critical rate-limiting steps. Even though reticulocytes could be produced in vitro, the low efficiency should be concerned. To uncover the molecular mechanism of erythroblast enucleation will facilitate ex vivo production of functional erythrocytes. This review summarizes three prevalent enucleation mechanisms, including apoptosis, asymmetric cytokinesis and vesicle trafficking, discusses proteins and microRNAs playing important role in the process, and evaluates the prospects for ex vivo production of red blood cells.