Endothelial to hematopoietic transition (EHT) is a dynamic process involving the shutting down of endothelial gene expression and switching on of hematopoietic gene transcription. Whilst the factors regulating EHT in hemogenic endothelium (HE) of the dorsal aorta have been relatively well studied, the molecular regulation of yolk sac HE remains poorly understood. Here, we show that SOX7 inhibits the expression of RUNX1 target genes in HE, whilst having no effect on RUNX1 expression itself. We establish that SOX7 directly interacts with RUNX1 and inhibits its transcriptional activity. Through this interaction we demonstrate that SOX7 hinders RUNX1 DNA binding as well as the interaction between RUNX1 and its cofactor CBFβ. Finally, we show by single cell expression profiling and immunofluorescence that SOX7 is broadly expressed across the RUNX1(+) yolk sac HE population compared with SOX17. Collectively, these data demonstrate for the first time how direct protein-protein interactions between endothelial and hematopoietic transcription factors regulate contrasting transcriptional programs during HE differentiation and EHT.