Organisms that reproduce sexually have evolved highly sophisticated mechanisms to generate two sexes. Some hymenopterans (such as ants, bees, and wasps) have a complementary sex-determination system in which heterozygosity at one CSD locus induces female development, whereas hemi- or homozygosity at the locus induces male development. This system can generate a high cost of inbreeding, as individuals that are homozygous at the locus become sterile, diploid males. On the other hand, some hymenopterans have evolved a multi-locus, complementary, sex-determination system in which heterozygosity in at least one CSD locus induces female development. This system effectively reduces the proportion of sterile diploid males; however, how multiple primary signals pass through a molecular cascade to regulate the terminal sex determination gene has remained unclear. To clarify this matter, we investigated the molecular cascade in the ant,Vollenhovia emeryi, with two CSD loci. Here we show thattransformer(tra) is necessary for proper feminization. Expression and functional analysis showed that individuals heterozygous in at least one of the two CSD loci develop into females. Individuals heterozygous at only a single CSD locus do not develop into sexual intermediates, because the signal derived from the heterozygous CSD is amplified by a positive-feedback splicing loop oftra. Our data also demonstrate that tra controls splicing ofdoublesex(dsx), which is involved in sexual differentiation. We suggest a cascade model to arrive at a binary determination of sex under multiple primary signals.