Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDRs and the precise positioning of the +1 nucleosomes are maintained on active genes remains unclear. Here, we report that the Drosophila nonspecific lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. Not only is the NSL complex essential for transcription, but it is required for accurate TSS selection for genes with multiple TSSs. Furthermore, loss of the NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.