Glucose levels influence HSC formation via increased energy metabolism. (A) Hexokinase inhibitors lonidamine (lonid; 10μM) and 3BP (20μM) decreased runx1/cymb staining and eliminated the effect of glucose on HSCs; addition of pyruvate (pyr; 1%) rescued the block in HSC formation elicited by lonid, but not 3BP, which inhibits both glycolysis and oxidative phosphorylation (n ≥ 30/tx). (B) Treatment with electron transport chain inhibitors potassium cyanide (KCN) (100μM) and OAA (50μM) decreased HSC gene expression and blocked the effect of glucose (n ≥ 50/tx). (C) Quantitative analysis of runx1 expression confirms the impact of hexokinase inhibition on mitigating the effect of glucose on HSCs (ANOVA, P < .05, n = 3). (D) Quantitative analysis of double-positive (DP) cells in the AGM by fluorescence microscopy of cmyb:eGFP;lmo2:dsRed transgenic embryos confirms the observed inhibitory effects of lonid and OAA in vivo (t test, *P < .01 vs con, **P < .001 vs gluc, n = 5). (E) The ability of OAA to eliminate the impact of glucose on runx1 expression was corroborated by qPCR (ANOVA, *P < .05, n = 3).