In healthy peripheral blood stem cell (PBSC) donors, rare cases of transient thrombocytopenia have been reported due to the administration of granulocyte colony-stimulating factor (G-CSF) or the apheresis itself. Meanwhile, differentiating pseudothrombocytopenia induced by anticoagulants is crucial, as it can result in false low platelet counts. This study aimed to investigate the causes of thrombocytopenia in healthy PBSC donors. We investigated PBSC donors who experienced thrombocytopenia during transplantation at St. Mary’s Hospital, Seoul. Three donors were identified and donor workup studies were within the normal limits. After G-CSF administration prior to transplantation, the donors experienced a significant reduction in platelet counts. Apheresis resulted in lower levels, yet platelet counts returned to normal levels approximately two weeks later. In donor 3, thrombocytopenia was seen during the donor workup and ethylenediaminetetraacetic acid (EDTA)-induced pseudothrombocytopenia was identified after the supplementation of amikacin. For donor 3, we investigated whether his recipient’s sample showed EDTA-induced pseudothrombocytopenia through a review of any platelet clumping in the apheresis product and the presence of immunoglobulin M (IgM) or IgG antiplatelet antibodies in the recipient’s peripheral blood. In conclusion, the risk of severe thrombocytopenia with G-CSF administration in PBSC donors should be considered and accurate differentiation of pseudothrombocytopenia is imperative.