The development of a complex organ requires the specification of appropriate numbers of each of its constituent cell types as well as the correct positioning of these cell types within the organ. Our previous work on Drosophila embryonic heart development had shown that the Forkhead (Fkh/Fox) transcription factors Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) both specify cardiac progenitors by regulating the expression of the fibroblast growth factor receptor Heartless and the Wnt receptor Frizzled and determine the correct number of different cardiac cell types by regulating the subsequent division of the cardiac progenitors through a Polo-dependent pathway. Here we show that CHES-1-like and jumu are also required for the correct positioning of these cardiac cell types: loss-of-function mutations in or RNA interference knockdown of either gene results in the misalignment and incorrect locations of both cardial and pericardial cells within a hemisegment. Since defective cardiac progenitor cell divisions in CHES-1-like and jumu loss-of-function mutants frequently result in individual hemisegments having different numbers of cardiac cells than their partner across the dorsal midline, we initially examined this asymmetry as a possible cause of incorrect positioning. Our statistical analyses revealed that steric constraints imposed by the differing number of heart cells in contralateral hemisgments are not sufficient to explain all of the observed defects in heart cell positioning: statistically significant increases in the number of incorrectly positioned cardiac cells are also observed in Fkh mutants compared with wild-type embryos when only members of contralateral hemisegment pairs having the same number of each cardiac cell type are compared. In order to identify the genetic subnetworks utilized by CHES-1-like and jumu to bring about correct positioning, we next compared genome-wide transcription expression profiles of purified mesodermal cells from wild-type embryos and embryos either lacking CHES-1-like or jumu or overexpressing either of these Fkh genes. Gene ontology (GO) enrichment analysis of differential expressed genes in these Fkh mutants or Fkh-overexpressing embryos suggests that CHES-1-like and jumu could be regulating components of at least two known pathways involved in proper cardiac cell positioning: one involving Uncoordinated5 and NetrinB, and the other involving heterotrimeric G proteins and septate junction proteins. We are experimentally testing these hypotheses at present.