In exploring the utility of double-stranded RNA (dsRNA) injections for silencing the PAR-domain protein 1 (Pdp1) gene in adult Drosophila, we noticed a dramatic loss of fat tissue lipids. To verify that our RNAi approach produced the expected Pdp1 knockdown, the abdominal fat tissues sections were stained with PDP1 antibodies. PDP1 protein immunostaining was absent in flies injected with dsRNA targeting a sequence common to all known Pdp1 isoforms. Subsequent experiments revealed that lipid staining is reduced in flies injected with dsRNA against Pdp1 γ (fat body specific) and not against Pdp1 ε (predominantly involved in circadian mechanisms). Drosophila PDP1 γ protein shows a high homology to mammalian thyrotroph embryonic factor (TEF), albumin D site-binding protein (DBP), and hepatic leukemia factor (HLF) transcription factors. In an in vitro model of drug- (olanzapine-) induced adiposity in mouse 3T3-L1 cells, the mRNA content of HLF but not TEF and DBP was increased by the drug treatment. A knockdown of the HLF mRNA by transfecting the cultures with HLF dsRNA significantly reduced their lipid content. Furthermore, the HLF RNAi prevented olanzapine from increasing the cell lipid content. These results suggest that the PDP1/HLF system may play a role in physiological and drug-influenced lipid regulation.