Despite its central importance in gene regulation, chromatin in mammalian cells remains relatively poorly understood-a predicament due to the paucity of robust genetic tools in mammals, the complexity of the chromatin remodeling machinery, and the dynamic properties of chromatin in vivo. Here we review recent developments in understanding endogenous mammalian gene regulation via the use of designed transcription factors (TFs). These include mutated forms of naturally occurring TFs that exhibit dominant-negative activity, and designed proteins with novel, predetermined DNA-binding specificities. Systematic targeting of designed TFs to particular promoters is helping to illuminate the complex rules that chromatin imposes on TF access and action in vivo. We evaluate the potential applications of these proteins as probes of mammalian chromatin-based regulatory pathways and their potential for the therapy of human disease, highlighting leukemia in particular.