Filamins are required for the generation of normal size platelets. (A-C) microscopic images of FlnAB_Low and Fln_Normal ESC/EB-derived cells during the process of proplatelet production. Panel A contains a fluorescent image of GFP-positive, FlnAB_Low proplatelets, with its corresponding phase-contrast image in panel B. Arrows depict the typical large swellings and shafts produced in the absence of filamin. Compare these structures with those present in GFP-negative, filamin-positive proplatelets (C), which have much thinner swellings and shafts, and numerous small proplatelets coming off of the thin shafts. By CD41 Day 6, the majority of proplatelets produced in the FlnAB_Low cultures had become GFP and shRNA-negative, and began to reexpress filamins. Platelets shed into the culture media were identified by GPIbβ expression and forward scatter. (D-E) FACS analysis of platelet size and relative expression of CD41 and GPIbβ in the FlnAB_Low, GFP-positive versus the Fln_Normal, GFP-negative population. Note that platelets derived from the proplatelets that remained FlnAB_Low are approximately twice the size of those that had become FlnAB_Normal.