Insight into the regulation of core transcription factors is important for a better understanding of the molecular mechanisms that control self-renewal and pluripotency of human ESCs (hESCs). However, the transcriptional regulation of NANOG itself in hESCs has largely been elusive. We established a NANOG promoter luciferase reporter assay as a fast read-out for indicating the pluripotent status of hESCs. From the functional cDNA screens and NANOG promoter characterization, we successfully identified a zinc finger transcription factor KLF4 and a homeodomain transcription factor PBX1 as two novel transcriptional regulators that maintain the pluripotent and undifferentiated state of hESCs. We showed that both KLF4 and PBX1 mRNA and protein expression were downregulated during hESC differentiation. In addition, overexpression of KLF4 and PBX1 upregulated NANOG promoter activity and also the endogenous NANOG protein expression in hESCs. Direct binding of KLF4 on NANOG proximal promoter and PBX1 on a new upstream enhancer and proximal promoter were confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assay. Knockdown of KLF4/PBX1 or mutation of KLF4/PBX1 binding motifs significantly downregulated NANOG promoter activity. We also showed that specific members of the SP/KLF and PBX family are functionally redundant at the NANOG promoter and that KLF4 and PBX1 cooperated with OCT4 and SOX2, and transactivated synergistically the NANOG promoter activity. Our results show two novel upstream transcription activators of NANOG that are functionally important for the self-renewal of hESC and provide new insights into the expanded regulatory circuitry that maintains hESC pluripotency.