Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producers during viral infections. Using shRNA-mediated knockdown in an immortalized hematopoietic stem and progenitor cell line (iHSPC), we identified Mef2c as a novel transcription factor essential for pDC development. Conditional knockout of Mef2c in bone marrow or dendritic cells (DCs) markedly reduced pDC generation. Interestingly, Mef2c-deficient pDCs exhibited hyper-responsiveness to TLR stimulation, with enhanced upregulation of activation markers and increased type I IFN production, revealing dual roles of Mef2c in pDC development and function. To investigate physiological antiviral contexts, we examined pDC responses during HSV-1 challenge using an in vitro stimulation platform. pDCs were more effectively activated by coculture with HSV-1-infected bystander cells (e.g., MEFs or NIH3T3 fibroblasts) than by direct viral exposure. In an NIH3T3-based coculture system, Mef2c-deficient pDCs showed heightened expression of activation markers (CD86, PD-L1, and Sca-1) upon viral challenge compared to controls. Together, these findings demonstrate that Mef2c negatively regulates pDC antiviral responses while promoting their development. Future studies will define how Mef2c deficiency impacts pDC antiviral immunity and clarify the underlying mechanisms.