In zebrafish, TATA-box-binding protein (TBP)-related factor 3, Trf3, is required for early development and initiation of hematopoiesis, and functions by promoting expression of a single target gene, mespa. Recent studies have shown that in murine muscle cells, TRF3 interacts with the TBP-associated factor TAF3. Here we investigate the role of Taf3 in zebrafish embryogenesis. We find that like Trf3-depleted zebrafish embryos, Taf3-depleted embryos exhibit multiple developmental defects and fail to undergo hematopoiesis. Both Trf3 and Taf3 are selectively bound to the mespa promoter and are required for mespa expression. Significantly, Taf3 interacts with Trf3 but not Tbp, and a Trf3 mutant that disrupts this interaction fails to support mespa transcription, early development, and hematopoiesis. Thus, a selective interaction between Trf3 and Taf3 is required for early zebrafish development and initiation of hematopoiesis. Finally, we provide evidence that TRF3 and TAF3 are also required for hematopoiesis initiation in the mouse.