Prolonged isolated thrombocytopenia is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is associated with a poor prognosis. This study aimed to investigate the pathogenesis of prolonged isolated thrombocytopenia (PT). We analysed the expression of CX3CR1 on CD4 and CD8 T cells in bone marrow (BM) and peripheral blood (PB) at +90 days from allo-HSCT recipients with or without PT by flow cytometry analyses. We then determined the megakaryocytes ploidy distributions, apoptosis rate and Fas expression of recipients with or without PT in vitro directly or after depleting CD8(+) T cells or adding purified autologous CD8(+) T cells to CD8(+) T-dep MNCs. We found that the percentage of CD8(+) T cells in BM was higher in the patients with PT than in the controls. The elevated expression of the CX3CR1 was associated with PT. There was a marked increase in the percentage of low ploidy megakaryocytes in the recipients with PT. The depletion of CD8(+) T cells increased the apoptosis of megakaryocytes and decreased the expression of Fas, which could be corrected by re-adding purified autologous CD8(+) T cells. The increase of CD8(+) T cells and CD8(+)/CX3CR1(+) T cells in BM at +90 days were independent risk factors for PT according to multivariate analysis. Our data implied that the recruitment of CD8(+) T cells into BM might explain the suppression of megakaryocyte apoptosis through the elevated expression of CX3CR1(+) in PT after allo-HSCT. CX3CR1 might be a novel treatment target in recipients with PT.