Mouse bone marrow and spleen cells formed pure or mixed colonies of up to 80 megakaryocytes in agar cultures after stimulation by medium conditioned by activated mouse lymphoid cells. Megakaryocytes were identified on the basis of their morphology, polyploid mitoses and DNA content, and high cytoplasmic content of acetylcholinesterase. Megakaryocyte colony-forming cells were relatively small with a peak sedimentation velocity of 4.2 mm/hr. Spleen, lymph node, and thymus cells produced the factor stimulating megakaryocyte proliferation after culture in medium containing 2-mercaptoethanol, with or without added mitogens or allogeneic spleen cells. Peak activity in conditioning medium was associated with the small lymphocyte fractions in mouse spleen.