We have identified a rare ( approximately 0.05-0.1%) population of cells (Thy-1(hi)CD16(+)CD44(hi)CD2(-)TCRalphabeta(-)B220(-)M ac-1(-)NK1. 1(-)) in the adult mouse bone marrow that generates CD4(+) and CD8(+) TCRalphabeta(+) T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2(-)CD16(+)CD44(hi)TCRalphabeta(-) to CD2(+)CD16(int/-)CD44(int/-)TCRalphabeta(-) cells, and a later transition to CD4(+)CD8(+)TCRalphabeta(+) double-positive T cells that rapidly generate the CD4(+) and CD8(+) single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2(+)TCRalphabeta(-) stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.