To become mature αβ T cells, developing thymocytes must first assemble a T cell receptor (TCR) β chain gene encoding a TCRβ chain that forms a pre-TCR. These cells then need to generate a TCRα chain gene encoding a TCRα chain, which, when paired with the TCRβ chain, forms a selectable αβ TCR. Newly generated VJα rearrangements that do not encode TCRα chains capable of forming selectable αβ TCRs can be excised from the chromosome and replaced with new VJα rearrangements. Such replacement occurs through the process of TCRα chain gene revision whereby a Vα gene segment upstream of the VJα rearrangement is appended to a downstream Jα gene segment. A multistep, gene-targeting approach was used to generate a modified TCRα locus (TCRαsJ) with a limited capacity to undergo revision of TCRα chain genes. Thymocytes from mice homozygous for the TCRαsJ allele are defective in their ability to generate an αβ TCR. Furthermore, those thymocytes that do generate an αβ TCR have a diminished capacity to be positively selected, and TCRαsJ/sJ mice have significantly reduced numbers of mature αβ T cells. Together, these findings demonstrate that normal T cell development relies on the ability of developing thymocytes to revise their TCRα chain genes. © 2005 by The National Academy of Sciences of the USA.