The use of platelet transfusion to ensure the recovery of thrombopoiesis in patients constitutes high-cost support. The identification and cloning of recombinant human thrombopoietin (TPO) and the development of efficient methods of purification of hematopoietic stem cells and progenitor cells have ameliorated the development of strategies of ex vivo expansion of megakaryocyte (MK) progenitor cells and mature MKs. Synergistic combinations of cytokines including TPO, interleukin (IL)-1, IL-3, IL-11, stem cell factor, and FLT-3 ligand induce the ex vivo expansion of colony-forming unit-MK progenitors and MKs from cytokine-mobilized peripheral blood cells, bone marrow, and cord blood CD34+ cells. Depending on the various culture conditions, i.e., combinations of growth factors, initial concentration of CD34+, serum or serum-free cultures, and/or oxygen tensions, the expansion-fold of MKs and their progenitor cells vary greatly. The clinical applications of the reinfusion of ex vivo-generated MK cells have been investigated successfully in cancer patients following high-dose chemotherapy. This review reports the latest information concerning ex vivo expansion of MKs and the current status of clinical trials.