Von Willebrand factor (VWF) is essential for adequate hemostasis, and VWF deficiency results in clinically severe bleeding. Using single-cell RNA sequencing, researchers attempted to find new regulators of VWF levels in endothelial colony-forming cells (ECFCs) (scRNA-seq). ECFCs were extracted from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were employed as an extra control cell line. Cells were examined for Weibel Palade body (WPB) content and VWF release. All cell lines were subjected to scRNA-seq to look for gene expression heterogeneity and potential VWF regulators. Candidate modifiers discovered by scRNA-seq were further described using small-interfering RNA (siRNA) assays to assess their impact on VWF. They discovered that ECFCs produced from patients with low VWF had changes in baseline WPB parameters as well as impaired VWF release. scRNA-seq studies of these endothelial cells indicated lower VWF transcription overall, mosaicism of VWF expression, and genes that are differently expressed in low VWF ECFCs and control endothelial cells (control ECs). A siRNA search for possible VWF modifiers revealed additional regulatory possibilities, one of which, FLI1, changes VWF transcriptional activity. Finally, when ECFCs from people with low VWF were compared to control ECs, they showed differences in VWF packing and release. VWF transcription was found to be altered using scRNA-seq, and siRNA screening identified many putative VWF regulators.