Abstract: The aim of this study was to reprogram fibroblasts cell by in vitro transcribed mRNA cocktail of Oct4, Sox2 and SV40 T-antigen, and provide a safe, nonintegrating strategy for somatic cell reprogramming. The recombinated mRNA transcription vectors including the above three induced facotrs genes were constructed and transcripted, respectively, then 5′UTR and 3′ UTR of β-globin gene were used for stabilizing in vitro transcribed mRNA following in vitro capping and poly(A) tailing. After mRNA transfection of 293 and IMR90 cells, immunocytochemistry, immunoflurescence and Real-Time PCR methods were used to detect gene expression, protein location and endogenous gene expression related to pluripotence. The results showed that target gene expressions could be detected in 293 and IMR90 cells and all expressed protein were localized properly in the nucleus. The specific expression of Oct4 and Nanog were increased in transfection cells. Endogenous Nanog expression was induced by mRNA cock-tail transfection. These findings indicate that mRNA of Oct4, Sox2 and SV40 T in vitro can coorperate to initiate cellular reprogramming.