To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which governs the ability of the organism to proliferate in the lung. The other was ace1, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the Δace1 mutant is due to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.Author summaryAlthough A. fumigatus causes the majority of cases of invasive aspergillosis, the function of most of the genes in its genome remains unknown. To identify genes encoding transcription factors that may be important for virulence, we used a NanoString nCounter to measure the mRNA levels of A. fumigatus transcription factor genes in the lungs of mice with invasive aspergillosis. The transcriptional profiling data indicate that the organism is exposed to nutrient limitation and stress during growth in the lungs, and that it responds by up-regulating genes that encode mycotoxins and secondary metabolites. In vitro, this response was most closely mimicked by growth in medium that was deficient in nitrogen, iron and/or zinc. Using the transcriptional profiling data, we identified two transcription factors that govern A. fumigatus virulence. These were RlmA, which is governs proliferation in the lung and Ace1, which controls the production of mycotoxins and secondary metabolites.