The CD4+CD8+ (double positive, DP) stage of thymic development is thought to be the earliest period that generates natural Foxp3+ regulatory T (Treg) cells important for the prevention of autoimmunity. However, we found that most Foxp3+ DP cells identified by routine flow cytometry represent doublets comprised of Foxp3- DP and Foxp3+ CD4+CD8- (CD4SP) cells. This was determined using analysis of flow cytometric height and width parameters, postsort contaminants, and thymocyte mixing studies. Temporal analysis of Treg cell development arising from bone marrow precursors in neonatal bone marrow chimeras suggested that Foxp3+ DP cells are not a major percentage of Foxp3+ thymocytes, and it supported the notion that most Treg cell development occurred at the immature HSAhigh CD4SP stage. Thus, these data demonstrate that the frequency of Foxp3+ cells generated at the DP stage is much smaller than previously recognized, suggesting that additional thymocyte maturation may be required to facilitate efficient induction of Foxp3.