Postacrosomal sheath WW domain-binding protein (PAWP) is a novel sperm protein identified as a candidate sperm-borne, oocyte-activating factor (SOAF). However, regulation of PAWP gene expression is poorly understood. Therefore, we examined the PAWP gene expression across different stages of mouse embryonic stem cell (ESC)-based spermatogenesis in vitro and compared this expression at different stages of mouse testis development in vivo. Expression of PAWP was also examined in mouse embryonic fibroblasts (MEF), Sertoli cell, and the NIH3T3 cancerous cell line. We used a transgenic mouse ESC line C57BL/6J expressing Stra8-EGFP that was plated in murine ESC medium. To induce differentiation, cells were cultured on gelatin-coated medium with Retinoic Acid (RA) treatment. We applied reverse transcription-PCR and real-time PCR to analyze the differential expression of PAWP mRNA during different stages of mouse ESC differentiation in vitro parallel with mouse testis development in vivo and in cell lines. We found that expression of PAWP is increased during testis development in vivo with greatest expression at postmeiotic phase. It is also highly expressed in mouse ESC-derived germ-like cells after 30 d of RA induction in vitro. PAWP is remarkably expressed in mouse ESC and NIH3T3 cell line. These results indicate that PAWP plays a role in spermatogenesis and germ cell development. Moreover, we suggest PAWP as one of the markers that could be looked in ESC studies as a confirmed testis-specific gene. We also suggest an additional possible role for PAWP in proliferation of cancerous cell in general.