SummaryRUNX1 (previously termed AML1) is a frequent target of human leukaemia‐associated gene aberrations, and it encodes the DNA‐binding subunit of the Core‐Binding Factor transcription factor complex. RUNX1 expression is essential for the initiation of definitive haematopoiesis, for steady‐state thrombopoiesis, and for normal lymphocytes development. Recent studies revealed that protein arginine methyltransferase 1 (PRMT1), which accounts for the majority of the type I PRMT activity in cells, methylates two arginine residues in RUNX1 (R206 and R210), and these modifications inhibit corepressor‐binding to RUNX1 thereby enhancing its transcriptional activity. In order to elucidate the biological significance of these methylations, we established novel knock‐in mouse lines with non‐methylable, double arginine‐to‐lysine (RTAMR‐to‐KTAMK) mutations in RUNX1. Homozygous Runx1KTAMK/KTAMK mice are born alive and appear normal during adulthood. However, Runx1KTAMK/KTAMK mice showed a reduction in CD3+ T lymphoid cells and a decrease in CD4+ T cells in peripheral lymphoid organs, in comparison to their wild‐type littermates, leading to a reduction in the CD4+ to CD8+ T‐cell ratio. These findings suggest that arginine‐methylation of RUNX1 in the RTAMR‐motif is dispensable for the development of definitive haematopoiesis and for steady‐state platelet production, however this modification affects the role of RUNX1 in the maintenance of the peripheral CD4+ T‐cell population.