Following the recent realization that TCRβ transgenes can severely inhibit the rearrangement of endogenous Vβ gene segments in the absence of pre-TCRα (pTα) chains, we tested whether the pre-TCR has an essential role in TCRβ allelic exclusion under more physiological conditions by analyzing TCR rearrangement in immature thymocytes by single-cell PCR. Our results in pTα⁺ mice are consistent with an ordered model of TCRβ rearrangement beginning on one allele and continuing on the other only when the first attempt is unsuccessful. By contrast, a higher proportion of thymocytes from pTα^−/− mice exhibited two productive TCRβ alleles. Thus, the pre-TCR–independent suppression of rearrangement by TCRβ transgenes represents a transgene artifact, whereas under physiological conditions the pre-TCR is essential for allelic exclusion.