Maladaptive signaling by pro-inflammatory cytokines (PICs) such as Tumor Necrosis Factor α (TNFα), Interleukin-1β (IL1β), and Interferon ɣ (IFNɣ), can activate downstream signaling cascades that are implicated in the development and progression of multiple inflammatory diseases. Despite playing critical roles in pathogenesis, the availability of in vivo models in which to model tissue-specific induction of PICs is limited. To bridge this gap, we have developed a novel multi-gene expression system dubbed: Cre-Enabled and Tetracycline-Inducible transgenic system for conditional, tissue-specific expression of Pro-Inflammatory Cytokines (CETI-PIC3). This binary transgenic system permits the stoichiometric co-expression of TNFα, IL1β, IFNɣ, and an H2B-GFP fluorescent reporter gene in a dose dependent manner. Furthermore, cytokine mis-expression is enabled only in tissue domains that can be defined by Cre recombinase expression. We have validated this system in zebrafish using an insulin:cre line. In doubly transgenic fish, quantitative real-time polymerase chain reaction (qRT-PCR) demonstrated increased expression levels of IFNɣ, IL1β, and TNFα mRNA. Moreover, specific expression in pancreatic β cells was demonstrated by both TNF-α immunofluorescence and GFP fluorescence. Cytokine-overexpressing islets elicited specific responses: β cells exhibited increased expression of genes associated with reactive oxidative species (ROS)- mediated stress and endoplasmic reticulum (ER) stress, surveilling and infiltrating macrophages were increased, and β cell death was promoted. This powerful and versatile model system can be used for modeling, analysis, and therapy development of diseases with an underlying inflammatory etiology.