Acute codepletion of NR4A1/3 leads to loss of quiescence and increased proliferation of HCSs. (A) Experimental design for tamoxifen-dependent depletion of Nr4a1 in CDKO mice, using 4 daily treatments with tamoxifen (Nr4a1^fl/fl; Nr4a3^−/−; Rosa26-ER^T2). (B) NR4A1 depletion efficiency measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in HSCs after 4 daily tamoxifen injections (n = 3). (C) Representative flow cytometry plots illustrating subfractionation strategy of the HSPC (LSK) compartment to identify HSCs and early progenitors (MPP, HPC1, HPC2), based on surface expression of CD150 and CD48.⁶⁵  (D) Quantitation of HSPC (left and center, n = 5 CDKO and 3 control) and lineage-restricted progenitor cell frequencies (right, n = 3) at 4 days after acute NR4A1/3 codepletion. (E) Bone marrow cellularity after acute NR4A1/3 codepletion (n = 3). (F) Frequency of HSPCs using CD34, Flt3, CD48, and CD150 markers (n = 4). For F, statistical significance was calculated between wild-type and CDKO samples. Results expressed as mean ± SD. *P ≤ .05; **P ≤ .01; ***P ≤ .001. CLP, common lymphoid progenitors (Lin⁻CD11c⁻CD27⁺IL7r⁺Flt3⁺Ly6d⁻); CMP, common myeloid progenitors (Lin⁻Sca1⁻Kit⁺CD16/32⁻CD34⁺); GMP, granulocyte-macrophage progenitors (Lin⁻Sca1⁻Kit⁺CD16/32⁺CD34⁺); MEP, megakaryocyte-erythrocyte progenitors (Lin⁻Sca1⁻Kit⁺CD16/32⁻CD34⁻).