TPO treatment and inhibition of TGF-β1 activity increase MK numbers in BM culture in vitro. BM from PF4Cre;Tgfb1^flox/flox or WT mice were cultured with and without TPO for up to 5 days. BM cells from WT mice were cultured with TPO in the presence or absence of anti–TGF-β1–neutralizing antibody (α-TGF-β1; 2 μg/mL; AF101; R&D Systems). (A-B) MK numbers (large cells indicated by yellow arrows) were counted manually under a light microscope. (C-D) MK numbers were counted by flow cytometry after staining with CD41 antibody, and nuclei were labeled with Hoechst. (D) Representative flow cytometric dot plot analysis of gating of MKs with double-positive CD41⁺Hoechst⁺. (E) Plot shows MK numbers counted for indicated conditions. FSC, forward scatter; SSC, side scatter.