Alternate forms of the PolII transcription initiation machinery have been proposed to play a role in selective activation of cell-type-specific gene expression programs during cellular differentiation. The cannonball (can) gene of Drosophila encodes a homolog of a TBP-associated factor (dTAF5) protein expressed only in spermatocytes, where it is required for normal transcription of genes required for spermatid differentiation. We show that Drosophila primary spermatocytes also express four additional tissue-specific TAFs: nht (homolog of dTAF4), mia (homolog of dTAF6), sa (homolog of dTAF8) and rye (homolog of dTAF12). Mutations in nht, mia and sa have similar effects in primary spermatocytes on transcription of several target genes involved in spermatid differentiation, and cause the same phenotypes as mutations in can, blocking both meiotic cell cycle progression and spermatid differentiation. The nht, mia, sa and rye proteins contain histone fold domain dimerization motifs. The nht and rye proteins interact structurally when co-expressed in bacteria, similarly to their generally expressed homologs TAF4 and TAF12, which heterodimerize. Strikingly, the structural interaction is tissue specific: nht did not interact with dTAF12 and dTAF4 did not interact with rye in a bacterial co-expression assay. We propose that the products of the five Drosophila genes encoding testis TAF homologs collaborate in an alternative TAF-containing protein complex to regulate a testis-specific gene expression program in primary spermatocytes required for terminal differentiation of male germ cells.