CD27 and CD28 have emerged as indicators demarcating the transition of thymocytes through β-selection. We found that CD28 exhibits a greater dynamic range of expression during this phase, thus it was employed to further parse the DN/CD44− compartment in order to assess IL-7 signaling during the β-selection process. Plotting CD28 versus CD25 expression revealed six DN/CD44− populations. OP9-DL1 stromal cell co-culture was used to demonstrate a developmental linkage from DN3a (CD25+CD28−/lo) to DN3b (CD25+CD28+) to DN3c (CD25intCD28+) to DN4a (CD25−CD28+) to double positive (DP) and showed the DN4b (CD25−CD28hi) and DN4c (CD25−CD28−/lo) populations to be inefficient in producing DP cells. Using CD69 as an additional marker to further parse the DN4a population, we found the pre-DP cells to be the CD44−CD25−CD28intCD69−CD4−/loCD8−/lo subset. Using this refined developmental scheme, IL-7Rα expression was found to be transiently up-regulated post-β-selection in the DN3b and DN3c subsets; however, this increase did not confer enhanced responsiveness over that observed in the DN3a population. CD28 messenger RNA expression was up-regulated in post-β-selected cells, whereas transcripts for CD27, IL-7Rα and Bcl-2 were lower than that observed in the DN3a population. This study refines the current thymocyte differentiation scheme to allow for more detailed evaluation of events controlling early T-cell development, specifically surrounding the β-selection checkpoint.