EKLF induction affects erythroid differentiation. FACS (A,B) and morphologic (C,D) assessment of cells derived from day 6 EBs or at 24 (Mkd7) or 48 (Mkd8) hours after placement on OP9 cells with thrombopoietin (TPO) with or without doxycycline. Data in panel A ares presented as the ratio of induced to uninduced Ter119⁺ levels from 4 independent experiments after subtracting negative controls for each sample using the Overton cumulative histogram subtraction algorithm⁴⁸  (thus the pretreated EBd6 ratio is one). Representative FACS data of CD71 and Ter119 in panel B are boxed according to R1-R5 categories of progressive erythroid maturation as described in Zhang et al⁴⁹  and schematized in the lower left panel, with the arrow showing the FACS pattern for differentiation as it proceeds from least to most mature. Numbers in each box indicate the percentage of cells within each category. Proerythroblasts (pro), basophilic erythroblasts (baso), polychromatophilic erythroblasts (poly), and megakaryocytes (meg) are indicated in panel C (May-Grunwald/giemsa stain). The frequency of megakaryocytic cells in the Mkd8 samples (C,D) was 29% (± 6%) in the absence of induction compared with 7% (± 1%) in the induced sample based on counts from 2 fields of 100 cells each from 2 independent experiments. Morphologic criteria for R2-R5 (D) were as described.⁵⁰  Error bars in panels A and D are SD.