The majority (∼70%) of postselection CD4⁺ single-positive (SP) thymocytes are CD8^loCD4^hi. These cells express very low levels of CD8, undetectable by flow cytofluorimetric (FCM) analysis, but sufficiently high to allow purification by panning. Unlike the fully mature CD8⁻CD4^hi thymocytes, which account for the remaining ∼30% of the SP CD4⁺ thymocytes, CD8^loCD4^hi cells are functionally immature and short-lived unless they receive an unidentified maturation signal from the thymus. In this study, we tested the hypothesis that this signal is provided by a T cell receptor (TCR)–major histocompatibility complex (MHC) class II interaction. Using intrathymic transfer, we show that the immature CD8^loCD4^hi cells could complete their intrathymic maturation and populate the peripheral lymphoid organs in the absence of MHC class II (and class I) molecules. Furthermore, in mice devoid of class II (and class I) molecules, the progeny of CD8^loCD4^hi cells was long-lived and functionally reactive to allogeneic class II molecules, although their numbers in the spleen and the mesenteric lymph node were ∼40–50% lower than those in class II⁺ mice 5 mo after transfer. Control experiments demonstrated that the surviving cells did not originate from the contaminating mature thymocytes. These results demonstrate that the final maturation, proliferation, and peripheral survival (up to 5 mo) of at least some postselection CD4⁺ SP cells do not require the TCR–MHC class II interaction. They also indicate that the TCR–MHC class II interaction(s) required for the intrathymic development of long-lived CD4⁺ SP cells occurs before the CD4^hi SP stage of development.