The HMG-box transcription factor LEF1 controls many developmentally regulated genes, including genes that activate expression of the T-cell antigen receptor alpha chain (TCR-alpha) in developing thymocytes. At least two distinct isoforms of LEF1 are expressed, resulting from variable inclusion of LEF1 exon 6; however, the expression pattern of these isoforms and mechanism of splicing regulation have not been explored. Here we demonstrate that inclusion of LEF1 exon 6 is increased during thymic development and in response to signaling in a cultured T-cell line in a manner which temporally correlates with increased expression of TCR-alpha. We further find that inclusion of exon 6 is dependent on the signal-induced increase in expression and binding of the splicing factor CELF2 to two intronic sequences flanking the regulated exon. Importantly, loss of exon 6 inclusion, through knockdown of CELF2 or direct block of the exon 6 splice site, results in reduced expression of TCR-alpha mRNA. Together, these data establish the mechanistic basis of LEF1 splicing regulation and demonstrate that LEF1 alternative splicing is a contributing determinant in the optimal expression of the TCR-alpha chain.