Background: Transduction of defined factors such as Oct3/4, Sox2, Nanog, and c-myc has successfully achieved pluripotency. However, during the generation process of induced pluripotent stem (iPS) cells, genetic manipulation of certain factors may cause tumorigenicity by random genomic integration and the use of oncogenes, which limits further clinical application. Accordingly, there is ongoing an extensive search for new technologies. Methods and Results: Previously, we found that the delivery of ES cell-derived proteins enables the reprogramming of adult fibroblasts (protein-iPS cells), converting them into pluripotent stem cells without the forced expression of ectopic transgenes. During the process, gene expression and epigenetic status were converted from somatic to ES-equivalent status. Protein-iPS cells were biologically and functionally indistinguishable from ES cells in vitro. Furthermore, Protein-iPS cells possess in vivo differentiation and developmental potentials. However, the efficiency to generate iPS by ES cell extract was not entirely satisfactory. Here, we demonstrated that protein derived from iPS cells, which were generated by ES cell extract or viral transduction of 4 factors, reprogrammed somatic cells to ES like cells and surprisingly the efficiency to form Oct4 positive colonies was remarkably improved compared to ES cell extract. Finally, we performed a tetraploid blastocyst complementation experiment, which is considered the most stringent functional assay of pluripotency. We confirmed fetal animals (E11.5) have been derived from protein-iPS cells. Conclusions and Significance: Our results provide an efficient and safe strategy for the reprogramming of somatic cells that can be used to facilitate pluripotent stem cell-based cell therapy.