To determine the clinical significance of a minor population of glycosylphosphatidylinositol-anchored membrane protein-negative (GPI-AP−) cells in acquired aplastic anemia (AA), we quantified CD55− CD59− granulocytes and red blood cells (RBCs) in peripheral blood from 122 patients with recently diagnosed AA and correlated numbers of GPI-AP− cells and responses to immunosuppressive therapy (IST). The highly sensitive flow cytometry detected 0.005% to 23.1% of GPI-AP− cells in 68.0% of AA patients. Sixty-eight of 83 (91%) patients with an increased ratio of GPI-AP− cells (PNH+) responded to antithymocyte globulin (ATG) + cyclosporin (CsA) therapy, whereas 18 of 39 (48%) without such an increase (PNH−) responded. Failure-free survival rates were significantly higher (64%) among PNH+ than PNH− patients (12%) at 5 years (Figure) although overall survival rates were comparable between the groups. Numbers of GPI-AP− and GPI-AP+ cells increased in parallel among most PNH+ patients who responded to IST, suggesting that these cells are equally sensitive to immune attack. These results indicate that a minor population of GPI-AP− cells represents a reliable marker of a positive IST response and a favorable prognosis among patients with AA. Furthermore, immune attack against hematopoietic stem cells that allows PNH clonal expansion might occur only at the onset of AA.