Megakaryocytopoiesis is the cellular process by which stem cells progress through commitment, proliferation and differentiation, leading to the production of platelets. In the mouse, this process is accomplished within the bone marrow (BM) and spleen microenvironment and is carried out by regulatory molecules and accessory cells, including macrophages, fibroblasts and endothelial-like cells. Previously, we demonstrated that specific macrophage depletion, using liposomal-encapsulated clodronate (LIP-CLOD), induced a rapid recovery of the platelet count in a mouse model of immune thrombocytopenia. We now show that LIP-CLOD treatment also provoked enhancement of both megakaryocytopoiesis and thrombocytopoiesis. In fact, a dose-dependent increase in the number of BM and spleen megakaryocytes was detected after treatment and this pattern correlated inversely to the macrophage count detected in these organs. Furthermore, the mice treated with the higher dose of LIP-CLOD showed signs of enhanced thrombopoiesis as they had an increased frequency of reticulated platelets and an improvement in the total platelet count 2 d later. In addition, the in vitro cytokine-induced megakaryocytopoiesis in BM and spleen cell cultures was significantly augmented in the presence of LIP-CLOD. Taken together, these results suggest that BM and spleen microenvironmental macrophages could be involved in the regulation of megakaryocyte and platelet production.