Dietary deficiency of Zn rapidly impairs T-cell development. Six- to 8-week-old female C57BL/6 mice were fed a normal or Zn-deficient diet for up to 8 weeks. (A) Total thymus cellularity from untreated mice or after 21 or 56 days of ZD (untreated, n = 24, combined from animals harvested alongside either day 21 or day 56 mice; day 21, n = 15 over 3 independent experiments; day 56, n = 10 over 2 independent experiments). (B) Concatenated flow cytometry plots displaying CD4 and CD8 expression in the thymus (plots were gated on viable CD45⁺ cells). (C) Total number of CD4⁻CD8⁻Thy1⁺ DN, CD3⁻CD4⁻CD8⁺ iSP, CD4⁺CD8⁺ DP, CD3⁺CD8⁻CD4⁺ SP4, or CD3⁺CD8⁺ CD4⁻ SP8 thymocytes from untreated mice or after 21 or 56 days of ZD (untreated, n = 24, combined from animals harvested alongside either day 21 or day 56 mice; day 21, n = 15 over 3 independent experiments; day 56, n = 10 over 2 independent experiments). (D) Multiparameter flow cytometry data from untreated mice or 21 days after ZD was placed in UMAP space and clusters were generated based on relative MFI of markers of thymocyte maturation (CD25, CD44, Thy1, CD4, CD8, CD3). (E) Concatenated flow cytometry plots showing Ki-67 expression in DN3, DN4, iSP, and DP thymocytes from untreated mice or after 21 or 56 days of ZD. (F) Concatenated flow cytometry plots showing EdU and DAPI expression in CD45⁺ events from untreated mice or after 21 days of ZD. (G) Proportion G0-G1, S, and G2 cells assessed with EdU/DAPI on CD45⁺ events from untreated mice or after 21 days of ZD (n = 10, combined from animals harvested over 3 independent experiments). (H) Proportion of EdU⁺ events (S phase) gated on CD4⁻CD8⁻Thy1⁺ CD25⁺CD44⁻ DN (DN3), CD4⁻CD8⁻Thy1⁺ CD25-CD44⁻ DN (DN4), CD3⁻CD4⁻CD8⁺ (iSP), CD4⁺CD8⁺ (DP) from untreated mice or after 21 days of ZD. (I) Lineage-depleted BM cells were isolated from untreated 6-week-old C57BL/6 mice and cocultured with OP9-DL1 cells for 30 days in the presence or absence of ZnSO₄ (10 µM added from day 0). Concatenated flow cytometry plots displaying CD4 and CD8 expression at day 30 (n = 5 independent experiments). Graphs represent mean ± standard error of the mean (SEM); each dot represents a biologically independent observation. *P < .05; **P < .01; ***P < .001.