DN thymocytes sequentially progress through DN1, 2, 3, and 4 developmental stages in FTOCs. Sorted DN1 (top row) and DN2 (bottom row) thymocyte populations were seeded into FTOCs and analyzed at either 9 or 20 days. Cells were stained for expression of CD4 and CD8 surface markers. CD4 and CD8 DN cells at day 9 of culture were further analyzed for CD44 and CD25 expression. Percentages represent cell number in each quadrant.