Murine megakaryocytes (MKs) are defined by CD41/CD61 expression and acetylcholinesterase (AChE) activity; however, their stages of differentiation in bone marrow (BM) have not been fully elucidated. In murine lineage-negative (Lin(-))/CD45(+) BM cells, we found CD41(+) MKs without AChE activity (AChE(-)) except for CD41(++) MKs with AChE activity (AChE(+)), in which CD61 expression was similar to their CD41 level. Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs could differentiate into AChE(+), with an accompanying increase in CD41/CD61 during in vitro culture. Both proplatelet formation (PPF) and platelet (PLT) production for Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs were observed later than for Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs, whereas MK progenitors were scarcely detected in both subpopulations. GeneChip and semiquantitative polymerase chain reaction analyses revealed that the Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are assigned at the stage between the progenitor and PPF preparation phases in respect to the many MK/PLT-specific gene expressions, including beta1-tubulin. In normal mice, the number of Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs was 100 times higher than that of AChE(+) MKs in BM. When MK destruction and consequent thrombocytopenia were caused by an antitumor agent, mitomycin-C, Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs led to an increase in AChE(+) MKs and subsequent PLT recovery with interleukin-11 administration. It was concluded that MKs in murine BM at least in part consist of immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs and more differentiated Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs. Immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are a major MK population compared with AChE(+) MKs in BM and play an important role in rapid PLT recovery in vivo.