BACKGROUND: Autologous fat grafting (AFG) has emerged as a highly sought-after plastic surgery procedure, although its success has been hampered by the uncertain fat survival rate. Current evidence suggests that adipose-derived stem cells (ADSCs) may contribute to fat retention in AFG. In previous studies, it was confirmed that thymosin beta 4 (Tβ4) could enhance fat survival in vivo, although the precise mechanism remains unclear. METHODS: ADSCs were isolated from patients undergoing liposuction and their proliferation, apoptosis, anti-apoptosis, and migration were analyzed under Tβ4 stimulation using cell counting kit-8, flow cytometry, wound healing assay, and real-time quantitative PCR. The mRNA levels of genes relating to angiogenesis and Hippo signaling were also determined. RESULTS: Tβ4 at 100 ng/mL (p-value = 0.0171) and 1000 ng/mL (p-value = 0.0054) significantly increased ADSC proliferation from day 1 compared to the control group (0 ng/mL). In addition, the mRNA levels of proliferation-associated genes were elevated in the Tβ4 group. Furthermore, Tβ4 enhanced the anti-apoptotic ability of ADSCs when stimulated with Tβ4 and an apoptotic induction reagent (0 ng/mL vs. 1000 ng/mL, p-value = 0.011). Crucially, the mRNA expression levels of angiogenesis-related genes and critical genes in the Hippo pathway were affected by Tβ4 in ADSCs. CONCLUSIONS: Tβ4 enhances adipose viability in AFG via facilitating ADSC proliferation and reducing apoptosis, and acts as a crucial positive regulator of ADSC-associated angiogenesis. Additionally, Tβ4 could be accountable for the phenotypic adjustment of ADSCs by regulating the Hippo pathway. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .