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. METHODS 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 .