Twist1 is a bHLH transcription factor that regulates cell proliferation, migration, and differentiation in embryonic progenitor cell populations and transformed tumor cells. While much is known about Twist1's function in a variety of mesenchymal cell types, the role of Twist1 in endocardial cushion cells is unknown. Twist1 gain and loss of function experiments were performed in primary chicken endocardial cushion cells in order to elucidate its role in endocardial cushion development. These studies indicate that Twist1 can induce endocardial cushion cell proliferation as well as promote endocardial cushion cell migration. Furthermore, Twist1 is subject to BMP regulation and can induce expression of cell migration marker genes including Periostin, Cadherin 11, and Mmp2 while repressing markers of valve cell differentiation including Aggrecan. Previously, Tbx20 has been implicated in endocardial cushion cell proliferation and differentiation, and in the current study, Tbx20 also promotes cushion cell migration. Twist1 can induce Tbx20 expression, while Tbx20 does not affect Twist1 expression. Taken together, these data indicate a role for Twist1 upstream of Tbx20 in promoting cell proliferation and migration and repressing differentiation in endocardial cushion cells during embryonic development.