Plasmacytoid dendritic cells (pDCs) are key players in antiviral immunity. In addition to massive type I interferon production, activated pDCs express the apoptosis-inducing molecule TRAIL, which enables them to clear infected cells that express the TRAIL receptors TRAIL-R1 and TRAIL-R2. In this study, we examined the molecular mechanisms that govern TRAIL expression in human pDCs. We identify NGFI-A-binding protein 2 (NAB2) as a novel transcriptional regulator that governs TRAIL induction in stimulated pDCs. We show with the pDC-like cell line CAL-1 that NAB2 is exclusively induced downstream of TLR7 and TLR9 signaling, and not upon type I IFN-R signaling. Furthermore, PI3K signaling is required for NAB2-mediated TRAIL expression. Finally, we show that TRAIL induction in CpG-activated human pDCs occurs through two independent signaling pathways: the first is initiated through TLR9 signaling upon recognition of nucleic acids, followed by type I IFN-R-mediated signaling. In conclusion, our data suggest that these two pathways are downstream of different activation signals, but act in concert to allow for full TRAIL expression in pDCs.