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The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome., Author Summary The genomes of complex organisms encompass hundreds of millions of base pairs of DNA, and regulatory molecules must distinguish specific targets within this vast landscape. In general, regulatory factors find target genes through sequence-specific interactions with the underlying DNA. However, sequence-specific factors typically bind only a fraction of the candidate genomic regions containing their specific target sequence motif. Here we identify potential roles for chromatin environment and flanking sequence composition in helping regulatory factors find their appropriate binding sites, using targeting of the Drosophila dosage compensation complex as a model. The initial stage of dosage compensation involves binding of the Male Specific Lethal (MSL) complex to a sequence motif called the MSL recognition element [1]. Using data from a large chromatin mapping effort (the modENCODE project), we successfully identify an active chromatin environment as predictive of selective MRE binding by the MSL complex. Our study provides a framework for using genome-wide datasets to analyze and predict functional protein–DNA binding site selection.