The challenge in the field of molecular self-assembly is that the outcome of these processes is not easily predicted a priori, rather, results of self-assembly processes are often rationalized after the fact. In this study, a systemic approach to self-assembly is taken; the chemical structure of the starting molecule is iteratively changed (adding, removing or substituting particular functional groups) and the resulting self-assembled structure is observed via scanning tunneling microscopy. The focus of this study is on the functional groups that can form directional interactions (hydrogen- and halogen-bonds). We observe a metastable cyclic pentamer for isatin (1H-indole-2,3-dione) with density functional theory providing support for a cyclic structure stabilized by both NH· · ·O and CH· · ·O hydrogen bonds between neighboring molecules. The CH· · ·O hydrogen bond is made between the 7-position proton acting as the hydrogen bond donor and the 3-position carbonyl as the hydrogen bond acceptor, and calculations indicate that the isatin pentamer structure is 17 kJ/mol more stable than the dimer on the per molecule basis. To probe the importance of the CH···O hydrogen bond in stabilizing the isatin pentamer, we compare to isatin derivatives: we replace the 3-position carbonyl with a methyl group (3-methyl 2-oxindole), the 7-position proton with a fluorine (7-fluoroisatin), systematically move the location of the hydrogen bond donor/acceptor by one position, (phthalimide), and remove of the primary hydrogen bond donor (1,2-indandione and 1,3-indandione). We show that cyclic pentamer formation is either altered or precluded as a result of these substitutions. To our surprise, substituting the 6-position with a bromine (6-bromoisatin) which is a position remote to the positions of the hydrogen-bond contacts, does not result in the formation of cyclic pentamers on the surface. A monolayer of 6-bromoisatin consists of almost entirely “zipper” dimer structures. Additionally, the importance of CH···O bonding in forming isatin pentamers is supported by electrospray ionization mass spectrometry (ESI-MS) measurements, which include a magic-number isatin pentamer peak. A mass spectrum of 6-bromoisatin also shows a relatively intense pentamer peak, whereas the other derivative molecules show little clustering under the same conditions. This work is significant in understanding the role that the position of the hydrogen- and halogen-bond donor/acceptor groups has on the resulting 2D supramolecular assemblies.