A new culture method for murine megakaryocytic colonies in vitro using glass-fiber filters is described in this reprot. Conditioned media were made from murine spleen cells (BALB/C mouse and C57 black mouse) and also from human spleen cells (splenectomy cases of hereditary spherocytosis and gastric cancer). A linear relationship between the number of seed cells and the number of colonies formed was found up to 3 .times. 105 cells/dish and this relationship indicated a clonal origin of the colonies. The best cloning efficiency for megakaryocytic colony formation was obtained when the culture medium contained 20% of conditioned medium prepared by the incubation of spleen cells at a concentration of 2 .times. 106 cells/ml with pokeweed mitogen. Megakaryocytic colonies first appeared by day 3 of culture and the maximum number of colonies was found on day 5. Human spleen cell conditioned media formed about 60 colonies/dish but murine one formed about 40 colonies/dish. The results suggest that human spleen cell conditioned media have a higher stimulating activity for murine megakaryocytic colony formation than murine conditioned media, and this method permits in situ colony assay and the preparation of permanent slides for megakaryocytic colony formation.