Assembly of TCRalpha and TCRdelta genes from the TCRalpha/delta locus is tightly controlled for the proper generation of alphabeta and gammadelta T cells. Of >100 shared variable gene segments in the TCRalpha/delta locus, only a few are predominantly used for the TCRdelta gene assembly, while most are for TCRalpha. However, the importance and mechanisms of the selective variable gene rearrangement for T cell development are not fully understood. We report herein that the development of a tissue-specific gammadelta T cell population is critically affected by recombination signal sequence-associated restriction on the variable gene usage for TCRdelta assembly. We found that the development of substitute skin gammadelta T cells in mice deficient of the TCRgamma3 gene, which is used in wild-type skin gammadelta T cells, was drastically affected by the strain background. A Vgamma2(+) skin gammadelta T cell population developed in mice of the B6 but not the 129 strain backgrounds, due to a difference in the rearrangement of endogenous Vdelta7(+) TCRdelta genes, which paired with the Vgamma2(+) TCRgamma gene to generate the Vgamma2/Vdelta7(+) skin gammadelta T cell precursors in fetal thymi of the B6 background mice. The defective TCRdelta rearrangement of the 129-"Vdelta7" gene was associated with specific variations in its recombination signal sequence, which renders it poorly compatible for rearrangement to Ddelta genes. These findings provide the first direct evidence that recombination signal sequence-associated restriction on the variable gene usage for TCRalpha/delta gene assembly plays an important role in T cell development.