Insulator binding proteins (IBPs) play an important role in regulating gene expression by binding to specific DNA sites to facilitate appropriate gene regulation. There are several IBPs in Drosophila, each defined by their ability to insulate target gene promoters in transgenic assays from the activating or silencing effects of neighboring regulatory elements. Of these, only CCCTC-binding factor (CTCF) has an obvious ortholog in mammals. CTCF is essential for mammalian cell viability and is an important regulator of genome architecture. In flies, CTCF is both maternally deposited and zygotically expressed. Flies lacking zygotic CTCF die as young adults with homeotic defects, suggesting that specific Hox genes are misexpressed in inappropriate body segments. The lack of any major embryonic defects was assumed to be due to the maternal supply of CTCF protein, as maternally contributed factors are often sufficient to progress through much of embryogenesis. Here, we definitively determined the requirement of CTCF for developmental progression in Drosophila We generated animals that completely lack both maternal and zygotic CTCF and found that, contrary to expectation, these mutants progress through embryogenesis and larval life. They develop to pharate adults, which fail to eclose from their pupal case. These mutants show exacerbated homeotic defects compared to zygotic mutants, misexpressing the Hox gene Abdominal-B outside of its normal expression domain early in development. Our results indicate that loss of Drosophila CTCF is not accompanied by widespread effects on gene expression, which may be due to redundant functions with other IBPs. Rather, CTCF is required for correct Hox gene expression patterns and for the viability of adult Drosophila.