WT BM cells’ response to α-KG treatment under physioxia and ambient air. (A) The box plots show the relative abundance of 2OG and 2HG levels in WT LSK-CD150^hi cells in physioxia and ambient air estimated by single-cell flux estimation analysis. P value calculated by Wilcoxon rank sum test. (B) Number of WT HSCs after a 6-hour incubation with α-KG under physioxia and ambient air. (C) Number of WT-HPCs after a 6-hour incubation with α-KG under physioxia and ambient air. (D) 5-hmC levels in BM cells treated with α-KG or DMSO under physioxia and air. Data are presented as mean ± standard error of the mean. *P < .05; **P < .01; ***P < .001; ****P < .0001 when analyzed by Wilcoxon rank sum test for (A) and by 2-way analysis of variance with a post hoc Tukey’s multiple comparison test for (B-D). (E) Graphical abstract of WT and Tet2^−/− cell behavior and underlying mechanism in physioxia and ambient air. Under physioxia, WT HSC pool is maintained as a result of diminished Tet2 activity, which is inhibited as a result of reduced levels of α-KG (Tet2 cofactor) and increased levels of 2-HG (Tet2 inhibitor). Under ambient air, WT HSC is induced to differentiate to MPPs by increased ROS levels, increased Tet2 activity, and activated p38 MAPK pathway. Tet2^−/− cells demonstrate resistance to extraphysiologic oxygen effect. 2HG, 2-Hydroxyglutarate; 2OG, 2-Oxoglutarate; DMSO, dimethyl sulfoxide; ns, not significant.