Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs,mir-7148, let-7b, andmir-223in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae aftermir-7148, let-7b, andmir-223knockdowns. These results suggestedmir-7148, let-7b, andmir-223are repressors for thrombocyte production. We then explored miRWalk database forlet-7bdownstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes,rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, andlbx1bthat encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes followinglet-7bknockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes,irf5, tgif1, irf8, cebpa, androrcathat showed thrombocytosis and one gene,ikzf1that showed thrombocytopenia. Furthermore, we tested whethermir-223regulates any of the above 13 transcription factors aftermir-223knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed withlet-7bknockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with bothlet-7bandmir-223. We also identified thattgif1, cebpa, ikzf1, irf5,irf8, andikzf1play a role in thrombopoiesis. Since theikzf1gene showed a differential expression profile inlet-7bandmir-223knockdowns but resulted in thrombocytopenia inikzf1knockdown in both adults and larvae we also studied anikzf1mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.