design: FACS-sorted primary HSCs were co-cultured with OP9-DL4 cells as previously described (Holmes and Zuiga-Pflcker, CSH Protocols, 2009). Briefly, sorted HSCs were resuspended in OP9 culture medium supplemented with 5 ng /mL Flt-3L (R&D Systems), 1 ng/mL IL-7 (PeproTech), and 2.5 g/mL Plasmocin Prophylactic (InvivoGen), then seeded onto 80%-90% confluent OP9-DL4 plates. HSCs were split onto fresh OP9-DL4 plates every 4 days via mechanical liberation (without trypsin). For tamoxifen-inducible deletion at day 4 of co-culture, (Z)-4-Hydroxytamoxifen (Sigma) dissolved in ethanol was added to the supplemented OP9 culture medium at 1 M for 36 hours. An equivalent volume of ethanol was added to the control samples. After 36 hours, HSCs were replated onto fresh OP9-DL4 plates. On Day 8 of co-culture, cells were mechanically liberated from co-culture dishes and differentiating T cell subsets were isolated via FACS. Live (DAPI-), lineage-negative (CD11b-, CD11c-, CD19-, GR-1-, Ter119-, NK1.1-, TCR-, Epcam-), DN (CD4-, CD8-) gated cells were sorted as DN1 (CD25-, c-Kit+), DN2a (CD25+, c-Kit+), and DN3 (CD25+, c-Kit-). For genetic perturbations, DN subsets were additionally gated for tdTomato or GFP based on Cre activity as reported by the Rosa26nTnG allele.   RNA-seq library preparation was performed by UChicago genomics core facility. Briefly: 50,000-100,000 primary thymocytes or DN cells from OP9-DL4 co-cultures were FACS-sorted directly into RULT lysis buffer from Qiagen RNeasy UCP Micro Kit and total RNA was extracted using manufacturers instructions. mRNA was enriched and RNA-seq libraries were constructed using the Illumina TruSeq Stranded mRNA Kit. Paired-end, dual-index sequencing was performed on the Illumina NovaSeq 6000 platform.