Significance Sex determination is a fundamental biological problem faced by all metazoans. To understand the sex determination pathway, it is important to identify all the genes involved in this process. In this study, we have identified a novel gene, spenito ( nito ), which is required for sex determination in Drosophila melanogaster . Loss of nito function in the soma transforms female tissues to male, and loss of nito function in female germ-line stem cells changes their sexual identity and prevents them from proper differentiation. We show that nito is a cofactor for Sex-lethal ( Sxl ) auto regulation, a process that remains an important textbook model for regulated alternative splicing.