In vitro, the TAF_II60 component of the TFIID complex contributes to RNA polymerase II transcription initiation by serving as a coactivator that interacts with specific activator proteins and possibly as a promoter selectivity factor that interacts with the downstream promoter element. In vivo roles for TAF_II60 in metazoan transcription are not as clear. Here we have investigated the developmental and transcriptional requirements for TAF_II60 by analyzing four independent Drosophila melanogaster TAF_II60 mutants. Loss-of-function mutations in Drosophila TAF_II60 result in lethality, indicating that TAF_II60 provides a nonredundant function in vivo. Molecular analysis of TAF_II60 alleles revealed that essential TAF_II60 functions are provided by two evolutionarily conserved regions located in the N-terminal half of the protein. TAF_II60 is required at all stages of Drosophila development, in both germ cells and somatic cells. Expression of TAF_II60 from a transgene rescued the lethality of TAF_II60 mutants and exposed requirements for TAF_II60 during imaginal development, spermatogenesis, and oogenesis. Phenotypes of rescued TAF_II60 mutant flies implicate TAF_II60 in transcriptional mechanisms that regulate cell growth and cell fate specification and suggest that TAF_II60 is a limiting component of the machinery that regulates the transcription of dosage-sensitive genes. Finally, TAF_II60 plays roles in developmental regulation of gene expression that are distinct from those of other TAF_II proteins.