CRLF3 deficiency causes ineffective thrombopoiesis. (A) Romanovsky-stained blood smear from Crlf3^−/− mouse whole blood taken at 100× magnification under light microscopy. (B) Crlf3^−/− blood smear stained with CD41 (green) and von Willebrand factor (VWF; red) and imaged by confocal microscopy. (C,D) Washed Crlf3^−/− platelets fixed and prepared for scanning (C) or transmission electron microscopy (D). Scale bars are 5 μm (B,C) and 2 μm (D). (E-G) Example flow cytometric plots (E) to determine platelet (GPV⁺/GPIIbIIIa⁺ events) (F) and preplatelet (GPV⁺/GPIIbIIIa⁺ events with larger forward scatter [FSC]/side scatter [SSC] than mature platelets) (G) counts from control (WT; blue) and Crlf3^−/− (red) splenectomized mice (n = 4 Crlf3^−/− postsplenectomy; n = 5 all other groups). (H) Quantification of MKs in hematoxylin and eosin–stained sections of control (WT; blue) and Crlf3^−/− (red) tibia of nonsplenectomized animals (left) or 21 postsplenectomy (right; n = 6 non-splenectomized and 2 splenectomized). Data represent means ± standard deviations (except splenectomized mice in panel H, where data represent means). Two-way analysis of variance with correction for multiple comparisons using the Holm-Sidak method (F,G) and unpaired 2-tailed Student t test (H). **P < .01, ***P < .005. FITC, fluorescein isothiocyanate; HPF, high-powered field; ns, not significant; PE, phycoerythrin.