Runx1/AML1 transcription factor expression in hematopoietic cell lineages is differentially regulated via usage of two distinct promoters. The 5' UTR and a 19 amino acid encoding sequence transcribed from the distal promoter is inserted via alternative splicing into the 5' end of the mRNA transcript, replacing the 5' UTR and a 5 amino acid encoding sequence usually transcribed from the proximal promoter. Expression of proximal Runx1 in 32Dcl.3 cells delays G-CSF induced neutrophil terminal differentiation by increasing viability compared to distal Runx1. We utilized Runx1 Nterminal deletion and point mutants of three evolutionarily conserved residues to describe dual N-terminal isoform motifs that promote two distinct differentiation phenotypes as regulatory elements in hematopoietic cell differentiation. Runx1 isoforms were evaluated in established hematopoietic in vitro and ex vivo differentiation systems. Deletion of amino acids 3’-14’ (Δ3-14) or 3’-19’ (Δ3-19) of the distal Runx1 N-terminus delayed terminal differentiation of the 32Dcl.3 myeloid cell line, indicating a regulatory motif in distal Runx1 abrogates the delay of terminal differentiation induced by proximal Runx1. Deletion of amino acids 3’-8’ (Δ3-8) or mutation of amino acids serine 3’, serine 5’ and phenylalanine 7’ of the distal Runx1 N-terminus reduce Runx1 expression in the 32Dcl.3 cell line. The N-terminus motif, runt domain and nuclear matrix-targeting sequence of Runx1 modulated Ets1 activity on the KIR3DL1 bidirectional promoter element. The transcription factor YY1 promotes both forward and reverse activation of the KIR3DL1 bidirectional promoter element dominantly in the presence of Runx1, and additively with Ets1. Distinct Runx1 proximal and distal N-termini induced phenotypes were observed in myeloid and thymocyte differentiation, but not with the KIR3DL1 luciferase assay system. This work identifies a previously unknown N-terminal regulatory motif that acts with spatio-temporal and gene target specificity to add another level of control over Runx1 activity during hematopoiesis.