Coordinate activation of transcription by multiple enhancer binding factors is essential for the regulation of pattern formation during development of Drosophila melanogaster. Cell-free transcription reactions are described that recapitulate transcriptional synergism directed by the Drosophila developmental regulators Bicoid (BCD) and Hunchback (HB). Within the basal transcription factor complex TFIID, two specific targets, TAFII110 and TAFII60, served as coactivators to mediate transcriptional activation by these two enhancer binding proteins. A quadruple complex containing TATA binding protein (TBP), TAFII250, TAFII110, and TAFII60 mediated transcriptional synergism by BCD and HB, whereas triple TBP-TAFII complexes lacking one or the other target coactivator failed to support synergistic activation. Deoxyribonuclease I footprint protection experiments revealed that an integral step leading to transcriptional synergism involves the recruitment of TBP-TAFII complexes to the promoter by way of multivalent contacts between activators and selected TAFIIs. Thus, the concerted action of multiple regulators with different coactivators helps to establish the pattern and level of segmentation gene transcription during Drosophila development.