An important site for bovine herpesvirus 1 (BoHV-1) latency is sensory neurons within trigeminal ganglia (TG). The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. Expression of four Krüppel-like transcription factors (KLF), KLF4, KLF6, PLZF (promyelocytic leukemia zinc finger), and KLF15, are induced in TG neurons early during dexamethasone-induced reactivation. The glucocorticoid receptor (GR) and KLF15 form a feed-forward transcription loop that cooperatively transactivates the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter that drives infected cell protein 0 (bICP0) and bICP4 expression. Since the bICP0 gene also contains a separate early (E) promoter, we tested the hypothesis that GR+KLF family members transactivate the bICP0 E promoter. GR+KLF4, both pioneer transcription factors, cooperated to stimulate bICP0 E promoter activity via a ligand independent manner in mouse neuroblastoma cells (Neuro-2A): furthermore, GR+KLF4 stimulated productive infection. Mutating both ½ GR response elements did not significantly reduce GR+KLF4 mediated transactivation of the bICP0 E promoter suggesting a novel mechanism exists for transactivation. GR+KLF15 cooperatively stimulated bICP0 activity less efficiently than GR+KL4: however, KLF6 and PLZF plus GR had little effect on the bICP0 E promoter. GR, KLF4, and KLF15 occupied bICP0 E promoter sequences in transfected Neuro-2A cells. GR and KLF15, but not KLF4, occupied the bICP0 E promoter at late times during productive infection of bovine cells. Collectively, these studies suggest cooperative transactivation of the bICP0 E promoter by two pioneer transcription factors (GR and KLF4) correlates with stimulating lytic cycle viral gene expression following stressful stimuli.