Significance MicroRNAs (miRNAs) are noncoding RNAs with diverse roles in development and pathogenesis. Biogenesis of canonical miRNA requires nuclear processing by DROSHA, nuclear export by Exportin 5, and cytoplasmic processing by DICER. To gain a deeper understanding of the maturation processes, we here ablated the DROSHA , Exportin 5 , and DICER genes using the same human cell line. Canonical miRNA production was abolished in DROSHA -deleted cells, revealing an irreplaceable role of DROSHA. Interestingly, however, some canonical miRNAs were still produced without DICER albeit at markedly reduced levels, and many were detected in Exportin 5 -deleted cells at only modestly decreased levels. Our study allows us to understand differential contributions of key biogenesis factors, and provides valuable resources for miRNA research.