Adipose-derived stromal cells (ASCs) possess significant regenerative potential, playing a key role in tissue repair and angiogenesis. During wound healing, ASC interacts with the extracellular matrix by recognizing arginylglycylaspartic acid (RGD) motifs, which are crucial for mediating these functions. This study investigates how RGD exposure influences ASC behavior, with a focus on angiogenesis. To mimic the wound-healing environment, ASC were cultured in a porcine gelatin sponge, an RGD-exposing matrix. Transcriptomics revealed that ASC cultured in gelatin exhibited an upregulated expression of genes associated with inflammation, angiogenesis, and tissue repair compared to ASC in suspension. Pro-inflammatory and pro-angiogenic factors, including IL-1, IL-6, IL-8, and VEGF, were significantly elevated. Functional assays further demonstrated that ASC-conditioned media enhanced endothelial cell migration, tubulogenesis, and reduced endothelial permeability, all critical processes in angiogenesis. Notably, ASC-conditioned media also promoted vasculogenesis in human vascular organoids. The inhibition of ASC-RGD interactions using the cyclic peptide cilengitide reversed these effects, underscoring the essential role of RGD-integrin interactions in ASC-mediated angiogenesis. These findings suggest that gelatin sponges enhance ASC’s regenerative and angiogenic properties via RGD-dependent mechanisms, offering promising therapeutic potential for tissue repair and vascular regeneration. Understanding how RGD modulates ASC behavior provides valuable insights into advancing cell-based regenerative therapies.