During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate γδ T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM-SAP signaling pathway regulates the development and function of multiple innate-like γδ T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent γδ TCR repertoire. SAP deficiency resulted in both a significant loss of an immatureGzma+Blk+Etv5+Tox2+γδT17 precursor population, and a significant increase inCd4+Cd8+Rorc+Ptcra+Rag1+thymic γδ T cells. SAP-dependent diversion of embryonic day 17 thymic γδ T cell clonotypes into the αβ T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered γδ TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset. Together, the data suggest that SAP-dependent γδ/αβ T cell lineage commitment regulates γδ T cell developmental programming and shapes the γδ TCR repertoire. Overall design: Single-cell CITE-Seq and TCR repertoire profiling of γδ T cells across various developmental stages and mouse models shows the influence of SLAM/SAP signaling on discrete γδ T cell developmental checkpoints and the shaping of the innate-like γδ TCR repertoire. Thymus single-cell CITE-seq with TCR repertoire analysis includes embryonic day 17 (E17) B6 and B6.Sh2d1a-/- (SAP-/-) mice with four biological replicates each, along with nine-day-old (D9) neonatal B6 and B6.SAP-/- mice with three biological replicates each. Additionally, the dataset encompasses thymus samples from six-week-old B6 and B6.SAP-/-mice with three replicates per group. Following thymic analysis, single-cell RNAseq with TCR repertoire analysis of adult lung γδ T cellsinvolved pooled cells from three 13-week-old B6 and B6.SAP-/- littermates per group. These experiments generated three libraries per experiments: RNA, VDJ, and cell surface protein (CSP). The cell surface protein (CSP) libraries encompassed both Antibody-derived tags (ADTs)and hashtag oligonucleotides (HTOs). This comprehensive approach sheds light on the molecular mechanisms underlying γδ T cell development and the impact of SLAM/SAP signaling across different developmental stages and mouse models. --------------------------------------- ADT/HTO barcode sequence hashtagged sample ADT sample TotalSeq-C0212 anti-mouse CD24 TATATCTTTGCCGCA E17thy, D9thy, W6thy TotalSeq-C0073 anti-mouse CD44 TGGCTTCAGGTCCTA E17thy TotalSeq-C1063 anti-mouse CD45RB TTGTATCTCCCTTGG E17thy TotalSeq-C0209 anti-mouse Vg1 TCGTTTAACCAGCCT E17thy TotalSeq-C0211 anti-mouse Vg4 AAGCTGCACCGTAAT E17thy, D9thy, W6thy, Lung TotalSeq-C0930 anti-mouse SLAMF6 CGATTCTTTGCGAGT E17thy, D9thy, W6thy TotalSeq-C0203 anti-mouse SLAMF1 CAACGCCTAGAAACC E17thy, D9thy, W6thy TotalSeq-C0077 anti-mouse CD73 ACACTTAACGTCTGG E17thy, D9thy, W6thy TotalSeq-C0301 anti-mouse hashtag 1 ACCCACCAGTAAGAC E17thy_B6-1, D9thy_B6-1, W6thy_B6-1 TotalSeq-C0302 anti-mouse hashtag 2 GGTCGAGAGCATTCA E17thy_B6-2, D9thy_B6-2, W6thy_B6-2 TotalSeq-C0303 anti-mouse hashtag 3 CTTGCCGCATGTCAT E17thy_B6-3, D9thy_B6-3, W6thy_B6-3, Lung_B6 TotalSeq-C0304 anti-mouse hashtag 4 AAAGCATTCTTCACG E17thy_B6-4, D9thy_SAPKO-1, W6thy_SAPKO-1, Lung_SAPKO TotalSeq-C0305 anti-mouse hashtag 5 CTTTGTCTTTGTGAG E17thy_SAPKO-1, D9thy_SAPKO-2, W6thy_SAPKO-2 TotalSeq-C0306 anti-mouse hashtag 6 TATGCTGCCACGGTA E17thy_SAPKO-2, D9thy_SAPKO-3, W6thy_SAPKO-3 TotalSeq-C0307 anti-mouse hashtag 7 GAGTCTGCCAGTATC E17thy_SAPKO-3 TotalSeq-C0308 anti-mouse hashtag 8 TATAGAACGCCAGGC E17thy_SAPKO-4, D9thy_ADTcontrol, W6thy_ADTcontrol