Prolonged isolated thrombocytopenia (PT) is a severe complication in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether the megakaryoctic potential of hematopoietic stem cells (HSCs) in bone marrow is intact and what factors drive the pathological process of PT remain elusive. A retrospective study in patients (n = 285) receiving HSCT revealed that the occurrence of PT was approximately 8% and the number of platelets and megakaryocytes in PT patients is much lower compared with control subjects. To test whether the deficiency of thrombopoiesis was caused by the activities of HSCs, the megakaryocytic differentiation potential of HSCs before or after transplantation was assessed. Interestingly, a substantial decrease of megakaryocytic differentiation was observed 2 weeks after transplantation of HSCs in all of the allo-HSCT recipients. However, 4 weeks after transplantation, the ability of HSCs to generate CD41⁺CD42b⁺ megakaryocytes in successful platelet engraftment patients recovered to the same level as those of HSCs before implantation. In contrast, HSCs derived from PT patients throughout the postimplantation period exhibited poor survival and failed to differentiate properly. A protein array analysis demonstrated that multiple inflammation-associated cytokines were elevated in allo-HSCT recipients with PT. Among them, insulin-like growth factor–binding protein 1 and regulated on activation, normal T cell expressed and secreted were found to significantly suppress the proliferation and megakaryocytic differentiation of HSCs in vitro. Our results suggested that the occurrence of PT may be attributed, at least partially, to the damage to HSC function caused by inflammation-associated cytokines after HSCT. These findings shed light on the mechanism underlying HSC megakaryocytic differentiation in PT patients and may provide potential new strategies for treating PT patients after HSCT.