The central nervous system’s (CNS) vast diversity of neural cells originates largely from regional specification of neural stem cell (NSC) phenotypes along the developing neural tube’s dorsoventral (D/V; stomach-to-back) and rostrocaudal (R/C, head-to-butt) axes. The morphogens that regulate regionalization along each axis are well established. However, how to apply the morphogens in vitro to precisely control NSC regionalization along the hindbrain and spinal cord’s R/C axis, which is governed by expression of 39 HOX genes, remained unknown. Using our ‘E6’ NSC derivation protocol, we discovered that temporal variations in the transition between a biphasic morphogen regimen permits deterministic patterning of HOX gene expression. Thus, scientists can now derive NSCs and differentiated phenotypes (e.g. motor neurons) regionalized to diverse, discrete rhombomere and vertebral domains. We’ve published these results in Stem Cell Reports, and our work vastly expands access to posterior CNS subtypes. In the future, we will use the protocol to investigate whether region-specifc neural cell transplants can regenerate functional spinal cord tissue after injury.