Foxp3 expression is a marker of regulatory T cells (T_reg), but how early it is expressed in the thymus is still not fully defined. In this study, we examined Foxp3 expression in double-negative (DN) CD4⁻CD8⁻ T cell precursors in the thymus by flow cytometry. By increasing the number of collected cells from the conventional 10⁴ cells up to more than 10⁶ cells during flow cytometry, we found that DN cells exhibited higher Foxp3 expression than double-positive (DP) CD4⁺CD8⁺ and single-positive (SP) CD4⁺ or CD8⁺ (SP) T cells. CD44⁺ expression positively correlated with Foxp3 in thymic DN cells. Furthermore, TCR-β⁻CD25⁺ DN cells exhibited the highest frequency of Foxp3-expressing cells. Almost all Foxp3⁺ cells expressed CD25in DN cells. These results suggest that Foxp3 expression in DN cells can directly be detected by flow cytometry and it was positively corelated with CD25 and CD44 in DN cells.