Objective <p>Acquired aplastic anemia (aAA) is characterized by an autologous immunological attack against hematopoietic stem and progenitor cells, and immunotolerance disruption is frequent, with reduced T regulatory cells (Tregs) frequencies and increased effector cytotoxic cells. Tregs are reduced in aAA and increase in number after successful immunosuppressive therapies.</p> Methods <p>In this retrospective study, we investigated the frequency of circulating Tregs by multiparametric flow cytometry immunophenotyping in non-severe aAA patients before and after immunosuppressive therapy. The samples were stained with the following antibodies: ECD anti-CD3, PE or PC5 anti-CD4, FITC anti-CD8, and PE anti-CD25, and Tregs were identified by first gating on linear parameters for lymphocyte identification and then for CD3 expression. In CD3⁺CD4⁺ cells, Tregs were further identified on the basis of CD25 and FOXP3 expression.</p> Results <p>Although the number of Tregs tended to increase after immunosuppressive treatments, their circulating frequency remained lower than that of healthy subjects, regardless of their responsiveness to therapies. Moreover, the relative frequency combined with absolute Treg counts might be more informative in the differential diagnosis of bone marrow failure syndromes.</p> Conclusions <p>The persistent decrease in circulating Tregs could be the result of immunosuppressive agents that could preferentially expand other T-cell subsets. At the same time, an imbalance in immunotolerance might persist, which is also favored by chronic antigen stimulation.</p>