Abstract Body: Up to 40% of arterial prosthetic bypass grafts (PG) will fail within 3 years, largely due to a fibroproliferative process called intimal hyperplasia (IH). Recently, attention has garnered for an “outside in” hypothesis whereby early expansion of adventitial mediators acts via paracrine effects to induce cellular change leading to IH. Activated adventitial fibroblasts (FB) and myofibroblasts (MFB) have been implicated in this process, though their role remains incompletely characterized. Integrated single-nuclei and spatial transcriptomics were used to analyze the prosthetic graft-arterial anastomosis of canine end-to-side carotid artery bypasses at 24h and 14d to elucidate pathways in FB activation and MFB differentiation. Clustering and differential abundance analysis demonstrated a spectrum of FB activation with distinct expression across timepoints and anastomotic location (Figure 1A). Activated FB populations expressing TGFB2 and GLIS3 emerged at 24h, while adventitial MFBs expanded at 14d. GLIS3⁺ and TGFB2⁺ FBs expressed differentiation and proliferation markers, suggesting a continuum of FB activation, whereas MFBs lacked such markers, suggesting terminal differentiation. Intercellular communication analysis revealed that MFBs express FN1 and SPP1, whose receptors were present on differentiating FBs and synthetic smooth muscle cells (s-SMCs). Trajectory analyses indicated that MFBs and endothelial-to-mesenchymal transition (EndMT) cells have a common lineage (Figure 1B), evidenced by shared MFB markers TIMP1 and VIM (Figure 1C), suggesting that MFBs may arise from both FB and endothelial cell (EC) precursors. Additionally, GLIS3⁺ FBs, TGFB2⁺ FBs, and MFBs all expressed high levels of TIMP1, favoring a secretory and proliferative phenotype and supporting MFB differentiation. Lastly, at 24h, IL-6 expression was prominent, primarily secreted by GLIS3⁺ FBs and s-SMCs, with IL-6 receptors on s-SMCs and ECs (Figure 1C-D). At 14d, FB-derived TGF-β expression predominated with TGF-β receptors on ECs and integrin aV receptors on FBs and SMCs (Figure 1C, E). In conclusion, a spectrum of early adventitial FB activation moving toward MFB differentiation occurs within a prosthetic graft-arterial anastomosis that is involved in inflammatory and pro-fibrotic signaling. Inhibition of this FB activation spectrum at the time of PG creation may be an attractive strategy to inhibit signaling cascades leading to downstream IH.