The fl (fluffy) gene of Neurospora crassa encodes a binuclear zinc cluster protein that regulates the production of asexual spores called macroconidia. Two other genes, acon-2 and acon-3, play major roles in controlling development. fl is induced specifically in differentiating tissue during conidiation and acon-2 plays a role in this induction. We examined the function of fl by manipulating its level of expression in wild-type and developmental mutant strains. Increasing expression of fl from a heterologous promoter in a wild-type genetic background is sufficient to induce conidiophore development. Elevated expression of fl leads to induction of development of the acon-2 mutant in nitrogen-starved cultures, but does not bypass the conidiation defect of the acon-3 mutant. These findings indicate that fl acts downstream of acon-2 and upstream of acon-3 in regulating gene expression during development. The eas, con-6, and con-10 genes are induced at different times during development. Morphological changes induced by artificially elevated fl expression in the absence of environmental cues were correlated with increased expression of eas, but not con-6 or con-10. Thus, although inappropriate expression of fl in vegetative hyphae is sufficient to induce conidial morphogenesis, complete reconstitution of development leading to the formation of mature conidia may require environmental signals to regulate fl activity and/or appropriate induction of fl expression in the developing conidiophore.