Abstract Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser 389in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser 389is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire. Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser389 in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser389 is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire. Supported by NIH R01 AI051454