Abstract Embryonic stem (ES) cells represent a popular model system for investigating development, tissue regeneration and repair. Although much is known about the molecular mechanisms that regulate the balance between self-renewal and lineage commitment in ES cells, the spatiotemporal integration of responsive signalling pathways with core transcriptional regulatory networks are complex and only partially understood. Moreover, measurements made on populations of cells reveal only average properties of the underlying regulatory networks, obscuring their fine detail. Here, we discuss the reconstruction of regulatory networks in individual cells using novel single cell transcriptomics and proteomics, in order to expand our understanding of the molecular basis of pluripotency, including the role of cell-cell variability within ES cell populations, and ways in which networks may be controlled in order to reliably manipulate cell behaviour.