The differentiation from multipotent hematopoietic stem cells (HSC) to mature and functional blood cells requires the finely tuned regulation of gene expression at each stage of development. Specific transcription factors play a key role in this process as they modulate the expression of their target genes in an exquisitely lineage-specific manner. A large number of important transcriptional regulators have been identified which establish and maintain specific gene expression patterns during hematopoietic development. Hematopoiesis is therefore a paradigm for investigating how transcription factors function in mammalian cells, thanks also to the evolution of genome-wide and the next-generation sequencing technologies. In this review, we focus on the current knowledge of the biological and functional properties of the hematopoietic master regulator RUNX1 (also known as AML1, CBFA2, PEBP2aB) transcription factor and its main downstream target PU.1. We will outline their relationship in determining the fate of the myeloid lineage during normal stem cell development and under conditions when hematopoietic development is subverted by leukemic transformation.