A. Simultaneous binding of multiple pioneer factors may be necessary for the formation of accessible chromatin at cis-regulatory elements. For example, during reprogramming of fibroblasts to iPSCs, Oct4, Sox2, and Klf4 are required to function together to define a subset of enhancers. B. Multiple pioneer factors (Zelda, GAGA factor, CLAMP, and Odd-paired (Opa)) function sequentially to regulate early embryonic development in Drosophila. C. Sequential action of pioneer factors can define cell-type specific enhancers that maintain gene expression during differentiation. As ESCs exit naïve pluripotency and become primed EpiLCs, GRHL2 defines novel enhancers to maintain expression of a gene regulatory network necessary for defining EpiLCs.