Cell fate determination relies on the ability of transcription factors (TFs) to select protein partners and specific regulatory elements to instruct a particular transcriptional output. Central to endothelial cell fate acquisition during embryonic development, the SOX18 transcription factor is a key regulator of both blood vascular and lymphatic endothelial cell specification. This TF is transiently expressed in all vascular beds during embryogenesis, however, the mechanisms that drive its specific molecular mode of action to instruct the differentiation of distinct sub-population of endothelial cells are currently unknown. To identify a global mechanism that could drive a differential activity of this TF in discrete cell subtypes, we analysed chromatin occupancy dynamics of SOX18 by combining Halo-tag technology with single molecule tracking in vitro. This approach identified that SOX18 binds to the chromatin via a two-component model; which means that SOX18 binds transiently to several non-specific sites, before binding more stably to specific target sites. Lastly, taking advantage of a non-functional SOX18 dominant-negative protein that causes the human syndrome Hypotrichosis-Lymphedema-Telangiectasia (HLT), we show that this non-functional TF interferes with the search pattern of SOX18 to identify its target genes. Our study shed light onto molecular behaviours of SOX18 TF on a genome-wide scale, and reinforces the concept that protein-protein interactions are central to govern target gene selectivity. We also uncover a global mechanism that explains at a molecular level how a dominant-negative protein disrupts TF activity and drives the aetiology a rare human vascular disease.