Abstract Developmental processes rely on collaboration of epigenetic modifications and sequence-specific transcription factors for timely activation and silencing of lineage-specific genes. Because histone H2A deubiquitination by 2A-DUB/Mysm1 was recently shown to affect hematopoiesis, in this investigation, we used 2A-DUB-deficient mice to systematically characterize the function of this epigenetic regulator in early T cell development. Thymi of 2A-DUB-/- mice were severely hypoplastic with >10-fold decrease in total cell numbers and increased fraction of apoptotic thymocytes compared with wild-type littermates. Diminished cell numbers resulted from a reduction of early thymocyte progenitors (ETPs) in context with defective HSCs, a partial block at the double-negative (DN)1-DN2 transition and increased apoptosis of double-positive (DP) thymocytes. In qPCR and IF analyses, we identified p19ARF and other genes involved in early T-cell development and protection from apoptosis as potential 2A-DUB target gene. ChIP analyses were conducted to explore binding of 2A-DUB to the Ink4a/Arf promoter in thymocytes. In addition, genetic approaches are used to validate the interaction of 2A-DUB/Mysm1 with known regulators of apoptosis and T cell development. We conclude that 2A-DUB/Mysm1 is critical for early T cell development via regulation of lineage-specific genes and that insufficient histone H2A deubiquitination induces apoptotic programs in HSCs, thymocytes and potentially other cell types.