High affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential of free fatty acid receptor 4 (FFA4) as a novel therapeutic target for the treatment of type II diabetes, the broad distribution pattern of this receptor suggests it may play a range of roles beyond glucose homeostasis in different cells and tissues. To date a single molecule, AH-7614, has been described as an FFA4 antagonist, however it's mechanism of antagonism remains unknown. We synthesized AH-7614 and two chemical derivatives and demonstrate these to be negative allosteric modulators (NAMs) of FFA4. Although these NAMs did inhibit FFA4 signalling induced by a range of endogenous and synthetic agonists, clear agonist probe-dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many anti-cancer drugs suggests that a novel close chemical analog of AH-7614 devoid of FFA4 activity, TUG-1387, will also provide a useful control compound for future studies assessing FFA4 function. Using TUG-1387 alongside AH-7614, we show that endogenous activation of FFA4 expressed by murine C3H10T1/2 mesenchymal stem cells is required for induced differentiation of these cells towards a more mature, adipocyte-like phenotype.