Pref-1/Dlk1 is made as an epidermal growth factor (EGF) repeat-containing transmembrane protein but is cleaved by tumor necrosis factor alpha converting enzyme (TACE) to generate a biologically active soluble form. Soluble Pref-1 inhibits adipocyte differentiation through the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and the subsequent upregulation of Sox9 expression. However, others have implicated Notch in Pref-1 signaling and function. Here, we show that Pref-1 does not interact with, or require, Notch for its function. Instead, we show a direct interaction of Pref-1 and fibronectin via the Pref-1 juxtamembrane domain and fibronectin C-terminal domain. We also show that fibronectin is required for the Pref-1-mediated inhibition of adipocyte differentiation, the activation of ERK/MAPK, and the upregulation of Sox9. Furthermore, disrupting fibronectin binding to integrin by the addition of RGD peptides or by the knockdown of alpha 5 integrin prevents the Pref-1 inhibition of adipocyte differentiation. Pref-1 activates the integrin downstream signaling molecules, FAK and Rac, and ERK activation by Pref-1 is blunted by the knockdown of Rac or by the forced expression of dominant-negative Rac. We conclude that, by interacting with fibronectin, Pref-1 activates integrin downstream signaling to activate MEK/ERK and to inhibit adipocyte differentiation.