Canalization, also known as developmental robustness, describes an organism's ability to produce the same phenotype despite genotypic variations and environmental influences 1,2. In Drosophila, Hsp90, the Trithorax group proteins, and transposon silencing have been implicated in canalization 3,4. Despite this, molecular mechanism underlying canalization remains elusive. Here, using an Drosophila eye-outgrowth assay sensitized by the dominant Kr Irregular facets-1 (Kr If-1), allele 3 , we show that the piRNA pathway, but not siRNA or miRNA pathways, is involved in canalization. Furthermore, we isolated a protein complex composed of Hsp90, Piwi, and the Hsp70/Hsp90 Organizing Protein Homolog (Hop), and demonstrated the function of this complex in canalization. Our data indicate that Hsp90 and Hop regulate the piRNA pathway via Piwi to mediate canalization. Moreover, they point to epigenetic silencing of the expression of existing genetic variants and the suppression of transposon-induced new genetic variation as two major mechanisms underlying piRNA pathway-mediated canalization. In both plants and animals, Hsp90 buffers against morphological changes induced either by genetic or non-genetic mechanisms, thereby promoting the robustness of the developmental programs that have been subjected to natural selection 5-8. However, under certain conditions, such as environmental stress, Hsp90 becomes overwhelmed, loosens its grip on canalization, and fails to repress the expression of genotype variants that have accumulated during evolution. The expressed phenotypes quickly become independent of Hsp90 deficiency, can be inherited in later generations, and could be subject to natural selection 3,5. In addition to Hsp90, maternally inherited epigenetic machineries also prevent expression of Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: