Kidney nephrons are composed of proximal and distal tubule segments that perform unique roles in excretion. The developmental pathways that establish nephron segment identities from renal progenitors are poorly understood. Here, we used the zebrafish pronephros to study nephron segmentation. We found that zebrafish nephron progenitors undergo elaborate spatiotemporal expression changes of many genes before adopting a segment fate. Initially, two domains of nephron progenitors are established, then are subdivided and demarcate individual nephron segments. Using genetic and chemical genetic models of retinoic acid (RA) deficiency, we discovered that RA modulates rostral progenitor formation. To delineate downstream pathways, we knocked down the irx3b transcription factor and found it regulates proximal tubule segment size and distal segment differentiation. Our results suggest a model whereby RA patterns the early field of nephron progenitors, with subsequent factors like irx3b acting to refine later progenitor subdomains and ensure activation of segment-specific gene programs. Developmental Dynamics 240:2011–2027, 2011. © 2011 Wiley-Liss, Inc.