Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators, is induced by cytokines that activate signal transducer and activator of transcription 5 (STAT5) and can inhibit STAT5 signaling in vitro. However, demonstration of a definitive in vivo role for CISH during development has remained elusive. This study employed expression analysis and morpholino-mediated knockdown in zebrafish in concert with bioinformatics and biochemical approaches to investigate CISH function. Two zebrafish CISH paralogues were identified, cish.a and cish.b, with high overall conservation (43-46% identity) with their mammalian counterparts. The cish.a gene was maternally derived, with transcripts present throughout embryogenesis, and increasing at 4-5 days post fertilization, while cish.b expression commenced at 8 hours post fertilization. Expression of cish.a was regulated by the JAK2-STAT5 pathway via conserved tetrameric STAT5 binding sites (TTCN₃GAA) in its promoter. Injection of morpholinos targeting cish.a – but not cish.b or control morpholinos – resulted in enhanced embryonic erythropoiesis, myelopoiesis and lymphopoiesis, including a 2-3 fold increase in erythrocytic markers. This occurred concomitantly with increased activation of STAT5. This study indicates that CISH functions as a conserved in vivo target and regulator of STAT5 in the control of embryonic hematopoiesis.