Mk lineage priming of HSCs is developmentally timed. (A) Gene set enrichment analysis comparing bulk RNA-sequencing results from sorted E14.5 FL and ABM HSCs (lineage^–Sca1⁺cKit⁺ [LSK] CD48^–CD150⁺Flt3^–) for the following lineage-restricted progenitor signatures^3,37: MkPs, pre-erythrocyte colony-forming units (preCFU-E), common lymphoid progenitors (CLP), and pre-granulocyte/macrophage progenitors (preGM). Normalized enrichment score (NES) and false discovery rate (FDR) are indicated. n = 2 to 3 biological replicates. (B) Normalized RNA-sequencing values for the average abundance of indicated transcripts per kilobase million. (C) Representative FACS analysis of CD41⁺ HSCs from E14.5 FL and ABM. (D) Mean frequency of CD41⁺ cells among HSCs from mice of the indicated ages. (E) Representative FACS analysis of MkPs in E14.5 and ABM. (F) Mean frequency of CD150⁺CD41⁺MkPs in Lin^–cKit⁺Sca1^– HPCs from mice of the indicated ages (n = 4-7). Statistical significance determined by Kruskal-Wallis test followed by Dunn’s multiple comparisons test comparing all ages vs E14.5 FL. (G) Representative images from day 7 single HSC cultures. (H) Analysis of day 7 single HSC cultures from the indicated ages. Wells with dead or no cells were removed before analysis (6%-8% of total wells). d, days; FSC, forward scatter; w, week(s).