Change of actin filament organization at the early stage of cell differentiation directs cell fate commitment of mesenchymal stem cells (MSCs). Thymosin beta-4 (Tbeta(4)), a major G-actin sequestering peptide, is known to regulate the cytoskeleton. The study investigated the ways in which Tbeta(4) regulates cell fate determination in MSCs upon differentiation induction. It was found that Tbeta(4) decreased F-actin formation, reduced the F-actin/G-actin ratio, and inhibited osteogenic differentiation; such actin reorganization was not associated with the change of Runt-related transcription factor 2 gene expression during early osteogenic induction. Besides, Tbeta(4) reciprocally facilitated adipogenic differentiation. Tbeta(4) treatment was found to up-regulate gene as well as promote surface expression of adipocyte adhesion molecule during early adipogenic differentiation, which accompanied acceleration of adipocyte phenotypic maturation but was not associated with differential expression of peroxisome proliferator-activated receptor gamma during the first week of adipogenic induction. In summary, Tbeta(4) initiated cell fate determination of MSCs through biophysical effects exerted by cytoskeleton reorganization and altered cell-cell adhesion rather than direct regulation of lineage-determining transcriptional factors. Such findings suggest that Tbeta(4), a ubiquitous peptide, may be involved in osteoporosis when its intracellular concentration is elevated. Further investigation of targeting Tbeta(4) for future osteoporosis treatment is warranted.