T-cell precursors that undergo productive rearrangements at the T-cell receptor (TCR) beta locus are selected for proliferation and further maturation, before TCRalpha expression, by signaling through a pre-TCR composed of the TCRbeta chain paired with a pre-TCRalpha (pTalpha) chain. Such a critical developmental checkpoint, known as beta-selection, results in progression from CD4(-) CD8(-) double negative (DN) to CD4(+) CD8(+) double positive (DP) TCRalphabeta(-) thymocytes. In contrast to mice, progression to the DP compartment occurs in humans via a CD4(+) CD8(-) intermediate stage. Here we show that the CD4(+) CD8(-) to CD4(+) CD8(+) transition involves the sequential acquisition of the alpha and beta chains of CD8 at distinct maturation stages. Our results indicate that CD8alpha, but not CD8beta, is expressed in vivo in a minor subset of DP TCRalphabeta(-) thymocytes, referred to as CD4(+) CD8alphaalpha(+) pre-T cells, mostly composed of resting cells lacking cytoplasmic TCRbeta chain (TCRbeta(ic)). In contrast, expression of CD8alphabeta heterodimers was selectively found on DP TCRalphabeta(-) thymocytes that express TCRbeta(ic) and are enriched for cycling cells. Interestingly, CD4(+) CD8alphaalpha(+) pre-T cells are shown to be functional intermediates between CD4(+) CD8(-) TCRbeta(ic)(-) and CD4(+) CD8alphabeta(+) TCRbeta(ic)(+) thymocytes. More importantly, evidence is provided that onset of CD8beta and TCRbeta(ic) expression are coincident developmental events associated with acquisition of CD3 and pTalpha chain on the cell surface. Therefore, we propose that the CD4(+) CD8alphaalpha(+) to CD4(+) CD8alphabeta(+) transition marks the key control point of pre-TCR-mediated beta-selection in human T-cell development.