Master differentiation transcription factors (MDFs) play decisive roles in cell lineage commitment and cellular functions. Forkhead box P3 (Foxp3) is the MDF essential for the lineage commitment and the suppression function of regulatory T cells (Tregs), which play critical roles in suppression of autoimmunity. Here, we analyzed transcription start sites (TSSs) and usage of the first exon of the mouse Foxp3 (mFoxp3) gene. In addition to known first exons -2a and -2b, we found a novel first exon, -2bΔ, which was the 3'-truncated form of the exon -2b. The major TSS of the exon -2bΔ was identical with that of the exon -2b. In contrast to the exon -2b and -2bΔ that have a major TSS, the exon -2a had multiple TSSs. Quantitative real-time RT-PCR revealed that the majority of mFoxp3 transcripts utilize the exon -2b as the first exon. Usage of the first exon of mFoxp3 was comparable in female and male Tregs. mFoxp3 transcripts with different first exons used the same downstream exons. Thus, selection of the first exons contributes to generation of diversity of mFoxp3 transcripts in mouse Tregs.