Development of FACS-based embryonic screening assays. (A) Schematic of hematopoietic development focusing on the circulating blood cell types and FL progenitor cell types assayed in our E14.5 screen. Early blood progenitors exit the posterior primitive streak (pps) and differentiate on the YS before entering the circulation from when the heart begins to beat at approximately E9.0. Large platelets also develop from the YS and enter the circulation early. Definitive HSCs enter and exit the midprimitive streak (mps) slightly later and migrate to the aorta-gonad-mesonephros (AGM) region where they develop with the ventral wall of the dorsal aorta where they lie dormant until the FL buds provides a receptive niche for HSC seeding and proliferation. In the FL, a hierarchy of progressively restricted progenitors is produced. Early in ontogeny, MEPs provide a high output of enucleated red cells and platelets. Later, myeloid progenitors generate neutrophils, and still later lymphoid progenitors (CLPs) seed the bone marrow and thymus where they provide B and T cells, respectively. We elected to screen the blood and FL at E14.5 so as to gain access to primitive and definitive blood cells (∼ 50:50 mix), and all definitive progenitor cell types. (B) The FL screen assays 6 functionally distinct HSC/progenitor cell subsets, LT-HSCs, MPPs, CLPs, CMPs, GMPs, and MEPs, by 8-color FACS analysis. (C) The FB screen assays 3 distinct subsets of megakaryo/erythroid cells, YS-derived primitive RBCs, FL-derived definitive RBCs, and platelets, by 3-color FACS analysis and cell size (on logarithmic scale). A representative plot of tissues from an E14.5 C57BL/6J embryo is shown, along with the percentage of nucleated blood cells (B) and percentage of total blood (C).