Thymosin β4 (Tβ4) was originally isolated as a thymic hormone from calf thymosin fraction 5 which exhibited both immune and endocrine functions in vivo and in vitro. Tβ4 is a ubiquitous peptide located in various tissues of mammalian species and other vertebrate classes. Recent studies on the molecular cloning and sequence analysis of rat and human Tβ4 cDNA have demonstrated that Tβ4 lacks a signal peptide which makes it unlikely that Tβ4 is a secretory peptide. The real function of Tβ4 is presently unknown, however, we focus this review on the molecular biology of Tβ4 with a special reference to the aspects of the Tβ4 gene expression in leukemic cells and cell lines during growth and differentiation.