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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 β1‐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.  Disclosure of potential conflicts of interest is found at the end of this article.

CD41 + /CD45 + Cells Without Acetylcholinesterase Activity Are Immature and a Major Megakaryocytic Population in Murine Bone Marrow