<p>Venous malformations (VMs) are developmental defects in vascular morphogenesis characterized by enlarged vein-like channels and defective perivascular cell coverage. The majority of VMs are caused by somatic mutations in endothelial cell (EC) receptor tyrosine kinase TIE2, but current medicinal options are limited. The common bottleneck when testing new therapeutic strategies and identifying efficient drug candidates is the absence of a reliable and robust model for replicating the characteristics of VMs. We report here on an advanced in vitro model for recapitulating cellular and molecular pathologies of the VM-causative <i>TIE2</i>^L914F mutation by co-culturing human adipose tissue-derived stem cells (hASCs) with either human umbilical vein ECs (HUVECs) retrovirally transduced to express <i>TIE2</i>^L914F or with induced pluripotent stem cell-derived ECs (iECs) with locus-targeted <i>TIE2</i>^L914F. For comparison, cellular and molecular crosstalk between vascular cell types was investigated in HUVEC and primary vascular smooth muscle cell (vSMC) co-cultures. Advanced microscopy and transcriptomic analysis were used to investigate the cellular and molecular phenotypes in vascular cell types. Comparison of <i>TIE2</i>^WT, gain- (<i>TIE2</i>^L914F) and loss-of-function mutations (<i>TIE2</i>^KO) revealed the importance of TIE2 signaling for vascular network formation in hASC/EC co-cultures. The model recapitulated the cellular hallmarks of the VMs and revealed gene signatures and signaling pathways relevant to VM pathologies. The data also demonstrated the utility of the hASC/EC model as a screening platform for VM pharmacotherapies. Overall, this work establishes a versatile platform for studying disease mechanisms affecting the vasculature and provides new insights into VM pathobiology and screening potential for therapeutic applications targeting VMs.</p>