Extracellular vesicles (EVs) are nano-sized lipid bilayer encapsulated particles with a molecular cargo that appears to play important roles within the human body, such as in cell-to-cell communication. Unraveling the composition of EV cargos remains one of the most fundamental steps toward understanding the role of EVs in intercellular communication and the discovery of new biomarkers. One of the unmet needs in this field is the lack of a robust, sensitive, and multiplexed method for EV mRNA profiling. We established a new protocol using the NanoString low RNA input nCounter assay by which the targeted mRNA transcripts in EVs can be efficiently and specifically amplified and then assayed for 770 mRNAs in one reaction. Prostate cancer cells with epithelial (PC3-Epi) or mesenchymal (PC3-EMT) phenotypes and their progeny EVs were analyzed by the same panel. Among these mRNAs, 157 were detected in PC3-Epi EVs and 564 were detected in PC3-EMT EVs. NOTCH1 was the most significantly abundant mRNA transcripts in PC3-EMT EVs compared to PC3-Epi EVs. Our results demonstrated that when cells undergo epithelial-to-mesenchymal transition (EMT), a more active loading of cancer progression-related mRNA transcripts may occur. The mRNA cargos of EVs derived from mesenchymal prostate cancer cells may contribute to the pro-EMT function. We found that mRNA transcripts are different in progeny EVs compared to parental cells. EV cargos are not completely reflective of their cell origin, and the underlying mechanism of cargo sorting is complicated and needs to be further elucidated.