The pre-T-cell receptor (TCR) is crucial for the early T-cell development, but the ligand for pre-TCR remains unidentified. We recently proposed a model that pre-TCR complexes oligomerize spontaneously through interactions of the pre-TCRα chain. To investigate the mechanism underlying this ligand-independent signaling in vivo, we established knock-in mice that express a pre-TCRα mutant lacking charged amino acids (D(22)R(24)R(102)R(117) to A(22)A(24)A(102)A(117); 4A). CD4(+)CD8(+) thymocyte number was significantly reduced in invariant pre-TCRα (pTα(4A/4A)) mice, whereas CD4(-)CD8(-) thymocytes were unaffected. The percentages of double-negative 3 (DN3) cells and γδ T cells were increased in the pTα(4A/4A) thymus, indicating that β-selection is impaired in pTα(4A/4A) mice. Pre-TCR-mediated tyrosine phosphorylation and clonal expansion into double-positive thymocytes were also defective in the knock-in mice. Pre-TCR was expressed at higher levels on pTα(4A/4A) cell surfaces than on those of the wild type, suggesting that the charged residues in pTα are critical for autonomous engagement and subsequent internalization of pre-TCR. Pre-TCR-mediated allelic exclusion of the TCRβ gene was also inhibited in pTα(4A/4A) mice, and thereby, dual TCRβs were expressed on pTα(4A/4A) T cells. Furthermore, the TCRβ chain variable region (Vβ) repertoire of mature T cells was significantly altered in pTα(4A/4A) mice. These results suggest that charged residues of pTα are critical for β-selection, allelic exclusion, and TCRβ repertoire formation.