Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.